[Frontiers in Bioscience 3, d944-960, September 1, 1998]
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BIOLOGICAL AND MOLECULAR BASIS OF HUMAN BREAST CANCER

Jose Russo, Xiaoqi Yang, Yun-Fu Hu, Betsy A. Bove, Yajue Huang, Ismael D.C.G. Silva, Quivo Tahin, Yuli Wu, Nadia Higgy, Abdel Zekri, and Irma H. Russo

Breast Cancer Research Laboratory, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

Received 12/17/97 Accepted 7/21/98

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Human Breast Epithelial Cells (HBEC) in Culture
4. Factors Influencing Susceptibility of HBEC to Cell Transformation
4.1. Lobular differentiation
4.2. Genetic predisposition
4.3. Cell immortalization
5. Molecular Mechanisms of Cell Immortalization
5.1. Activation of telomerase
5.2. Abrogation of cell cycle control
5.3. Genes preferentially expressed during cell immortalization
6. Molecular Mechanisms of Cell Transformation
6.1. Epigenetic mechanisms
6.2. Genetic mechanisms
7. Genomic changes in Immortalization and Transformation of HBEC
7.1. Genomic changes in cell immortalization
7.2. Genomic changes in cell transformation
8. Genomic Changes in Human Breast Lesions
9. Functional Roles of Chromosomes 11 and 17 in Transformed Phenotype Expression of HBEC
10. Summary and Perspectives
11. Acknowledgments
12. References

1. ABSTRACT

Human breast cancer remains the most common malignancy in the American women. The ultimate cure of this disease relies on a better understanding of the mechanisms underlying the initiation and progression of this disease. The neoplastic transformation of HBEC in vitro represents a successful model for obtaining knowledge on the molecular and biological alterations that may contribute to the tumorigenic mechanisms. We have presented here a current understanding of chemically transformed HBEC in the following aspects: 1. Factors affecting the transformation of HBEC such as genetic predisposition and differentiation status and prior immortalization; 2. New targets for studying the mechanism of cell immortalization such as alterations in telomerase activity and differential expression of cell cycle dependent genes as well as others recently isolated through differential cloning such as H-ferritin, and a calcium binding protein; 3. Epigenetic and genetic mechanisms underlying cell transformation; 4. The association of microsatellite instability in specific loci on chromosomes 11, 13, and 16 with the progression of cell transformation; and 5. The application of microcell mediated chromosome transfer technique as an approach to testing the functional role of specific genes whose dysregulation or loss of function may contribute to the ultimate cell transformation. Further efforts in this cell system will be directed to determine the roles of identified molecular changes as well as the mapping/cloning of tumor suppressor or senescence genes such as those that may reside on chromosomes 11 or 17.

2. INTRODUCTION

Breast cancer is the most common neoplastic disease in women with 180, 200 new cases reported annually (1). In spite of earlier detection and better treatment largely due to recent technologic advances, breast cancer is still the second leading cause of cancer-related death claiming 46, 000 lives of American women annually (2). Progress has been made in defining some of the critical processes associated with the development of breast cancer. However, the specific biochemical and molecular mechanisms underlying many of these complex carcinogenic events still remain to be elucidated. It is generally accepted that malignant transformation involves genetic and epigenetic changes that derail common regulatory mechanisms and result in uncontrolled cellular proliferation and/or aberrant programmed cell death or apoptosis (3, 4). These cellular abnormalities, hallmarks of a carcinogenic process, are frequently associated with molecular alterations involving activation of proto-oncogenes and inactivation of tumor suppressor genes as a result of genetic predisposition and/or exposure to physical (e.g., radiation), chemical (e.g., carcinogens, dietary components) and biological (e.g., viruses) environmental factors (5). A central challenge for cancer biology is to understand the cellular and molecular processes that drive normal human breast epithelial cells (HBEC) to neoplastic growth.

The vast majority of breast cancers are carcinomas, the malignant tumors of the epithelia. Based upon histological evaluations, development of breast cancer has been postulated to be a multi-step process and follows a defined sequence of qualitatively different events (6, 7) (Figure 1), as documented for a number of other malignancies (8, 9). In human breast, ductal hyperplasia and atypical ductal hyperplasia represent the initial stages of neoplastic growth and progress gradually to ductal carcinoma in situ, invasive ductal carcinoma and ultimate metastasis, even though normal cells could directly give rise to ductal carcinoma in situ or invasive ductal carcinoma (6, 10; Figure 1).

Figure 1. In vivo model of neoplastic progression of human breast carcinomas.

Analysis of pure populations of HBEC at various stages en route to malignancy would be the direct approach to understanding the cellular and molecular processes of breast carcinogenesis. However, primary cultures of HBEC from breast tissues at various neoplastic stages have been extremely difficult to establish and no cell lines at the intermediate stages of neoplastic transformation are available for mechanistic studies. The reasons for difficulties in establishing primary cultures of neoplastic breast cells are not fully understood and further refinements in culture techniques are clearly warranted for this experimental approach to be useful. On the other hand, primary culture of normal HBEC has been made possible by recent advances in tissue culture and, in some cases, normal HBEC lines have been successfully established (6, 11-14). Unlike rodent cells that are commonly used for oncogenic studies, normal HBEC do not exhibit spontaneous transformation in vitro and thus experimentally-induced transformation of normal HBEC in vitro has become a system of choice to elucidate the mechanism of breast carcinogenesis.

3. HUMAN BREAST EPITHELIAL CELLS IN CULTURE

It has been shown that the life span of HBEC cultured in adequate medium is comparable to that of adult human fibroblasts (30-40 doublings) (15, 16) and is profoundly affected by the concentration of calcium (Ca++) in culture medium (13). Extended growth of HBEC for over 1000 days and more than 50 generations without expressing terminal differentiation has been maintained by lower Ca++ level in culture medium (13). HBEC cultured under low-Ca++ conditions maintain their normal diploid karyotype, form domes and duct-like structures in collagen, express specific keratin filaments and milk fat globule membrane antigen, and contain all the other structural features of breast epithelial cells (17-19). In addition, a higher number of doublings has been observed in HBEC derived from breast tissues with a lower differentiation grade and a higher proliferation rate (6), indicating that the growth characteristics of HBEC in primary culture reflect the in vivo degree of lobular development and the rate of cell proliferation in vitro (6).

Like all normal diploid and differentiated somatic cells, normal HBEC have a limited capacity to divide both in vivo and in vitro. Cellular mortality of normal HBEC is characterized by a progressive cessation of cell growth manifested in cell culture by senescence that typically occurs after 10-20 passages in vitro (approximately 100 to 200 population doublings) (13, 16, 20). In contrast, transformed or tumor cells are able to escape from senescence as a result of genetic and epigenetic changes that disrupt the regulatory mechanisms of limited growth potential and are thereby considered immortal (21). Induction of immortality or immortalization involves abrogation of cellular programs for limiting the rate and the number of cell replication (22) and is generally perceived as the key event of an oncogenic process (13, 23).

Spontaneous immortalization of HBEC is a rarely-occurring event. Numerous investigators have tried to induce immortalization of HBEC using various physical (e.g., radiation), chemical (e.g., benzo(a)pyrene) and biological (e.g., viruses, gene transfer) approaches, the last being the most consistent (20). Immortalization of HBEC has been successfully induced by introduction of the human papilloma virus 16 (HPV-16) oncogenes E6 and/or E7 (24-26), simian virus 40 (SV40) DNA (27). However, immortalization of HBEC experimentally induced with viral oncogenes is often accompanied by expression of phenotypes indicative of neoplastic transformation, such as an increase in anchorage-independent growth and tumorigenesis in nude mice (27, 28). Clearly, a normal HBEC without expressing any transformed phenotypes is essential to any studies on experimentally-induced cell transformation.

We have reported that a mortal HBEC line #130, when cultured in medium containing 0.04 mM Ca++ (low Ca++) for over 2 years, became immortalized spontaneously giving rise to MCF-10F (12, 17). Immortalization of these cells is characterized by their continuous growth in culture medium containing the conventional level of Ca++ (1.05 mM; also called high Ca++) without entering senescence and expressing transformed phenotypes such as colony formation in agar or in agar-methocel (12, 13). Both the mortal and immortal cells are bona fide normal HBEC in nature, expressing genetic, cytogenetic, ultrastructural and phenotypic characteristics of normal human breast epithelia (12, 13) and represent cell lines closest to normal HBEC available.

The availability of normal HBEC in primary cultures, the mortal HBEC line (e.g., #130) and the immortal HBEC line MCF-10F provides us with a unique system to address some of the most important questions concerning the molecular mechanisms of cell transformation, such as: 1. What are the factors influencing susceptibility to cell transformation? 2. What are the molecular mechanisms responsible for cell immortalization? 3. What are the mechanisms associated with cell transformation?

4. FACTORS INFLUENCING SUSCEPTIBILITY OF HBEC TO CELL TRANSFORMATION

4.1. Lobular differentiation

Using our established in vitro system that allows us to efficiently culture HBEC whose growth characteristics in vitro reflect the in vivo degree of lobular development and the rate of cell proliferation (6), we tested on HBEC the transforming potential of chemical carcinogens known to be of etiological importance in various experimental models of mammary cancer (29). Our results indicate that HBEC of Lob 1, obtained from young nulliparous women, which are less differentiated, are more proliferative and more susceptible to be transformed by chemical carcinogens than those of the more differentiated Lob 3 from older and parous women (6). These results indicate that the susceptibility of HBEC in vitro is influenced by differentiation status of the breast in vivo. Whereas we do not have a complete elucidation of the genes involved in the process of mammary gland differentiation, we have recently cloned a gene, namely, mammary-derived growth inhibitor (MDGI), from the primary culture of HBEC (30). Using in situ hybridization techniques, we have demonstrated that the expression of MDGI is absent in the least differentiated Lob 1 and 2, low in the moderately differentiated Lob 3 and maximal in the most differentiated Lob 4 (30) (Table 1). In consideration of other observations that the MDGI locus, mapped to chromosome 1p21-23 (31), is frequently lost in breast tumors (32) and that MDGI expression is associated with tumor suppression in breast cancer cells (33), it is reasonable to speculate that MDGI might be a tumor suppressor and silenced in the very early stage of carcinogenesis. Overexpression of MDGI in the otherwise susceptible HBEC (e.g., MCF-10F cells) would confer resistance to chemical carcinogenesis in these cells. Experiments are currently in progress to test this hypothesis.

Table 1. Expression of MDGI in relation to lobular differentiation in human breast

Case #

Agea

Lobule Typeb

MDGIc

7

46

1

+/-

8

48

1

+/-

10

39

1

+/-

6

49

1/2

+

9

47

1/2

+/-

2

36

2

+

5

41

2/3

+

1

43

3

++

3

29

4

++++

4

35

4

++++

aAge of the donor at the time of reduction mammoplasty surgery.

bLobules type 1, 2, 3, and 4, classified according to histopathogical criteria described previously (11).

cIn situ hybridization with the antisense riboprobe of human MDGI. The intensity of the hybridization signal was scored as negative (-), weakly positive (+), moderately positive (++) and strongly positive (+++/++++).

4.2. Genetic predisposition

The primary cultures obtained from women with no family history of breast cancer, when treated with the carcinogens in vitro, exhibit an increase in survival efficiency (11, 13), which is perceived to precede the acquisition of anchorage independence (34, 35). In contrast, treatment of HBEC from women with family history of breast cancer with the same carcinogens induces the formation of colonies in agar-methocel (14) (Figure 2). Colonies formed from the treated cells showed considerable anchorage-independent growth during the 21-day assay period (Figure 3). However, when individual colonies were isolated, they failed to grow. Since formation of colonies in agar is generally construed as indicating anchorage-independent growth, a hallmark of neoplastic cells, our results clearly showed that HBEC from women with familial history of breast cancer manifested phenotypic changes indicative of initial stages of neoplastic transformation in response to the carcinogen treatment. Since inherited and acquired (spontaneous and induced) genetic changes can predispose an individual to both premalignant and malignant transformation of a specific organ (36, 37), it is reasonable to speculate that genetic predisposition in women with familial history of breast cancer may confer inherited susceptibility to environmental chemical carcinogens.

Figure 2. Survival and colony efficiencies in agar methocel of chemical carcinogen-treated HBEC in vitro (Reproduced with permission from Ref. 11).

Figure 3. Colonies formed in agar methocel in chemical carcinogen-treated HBEC from women with familial history of breast cancer (Reproduced with permission from Ref. 11).

4.3. Cell immortalization

Since in vitro treatment of HBEC in primary cultures with chemical carcinogens failed to induce the full expression of malignant transformation, we decided to use the protocols developed for primary culture of HBEC with the immortalized cell line MCF-10F in order to elucidate whether immortalization is required for the expression of the full malignant phenotype. Upon treatment of MCF-10F cells with 7, 12-dimethylbenzo[a ]anthracene (DMBA), N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (NMU), or benzo[a ]pyrene (BP), the treated cells showed altered morphology and altered pattern of growth, increased growth rate and anchorage independent growth in agar-methocel after 8-10 passages (around 157 days after the treatment). Control cells never developed colonies, whereas carcinogen-treated cells formed colonies from which the following clones were derived: D1, D2, and D3, from DMBA, M4 from MNNG, and BP1, BP2, BP5, BP6, BP7, and BP10 from BP treated cells (38) (Figure 4). All clones grew at a faster rate than their respective parental cells. Based upon this growth advantage, selected cell populations of clones BP1 and BP2 were isolated at approximately 446 days post-treatment; they were named BP1E and BP2B, respectively. BP1 and BP1E cells showed an increase in anchorage-independent growth, chemotaxis and invasiveness. From the D3 clone the D3-1 cell line was originated. It showed increased chemotactic and invasive capabilities, but to a lesser degree than BP1E. The tumorigenic potential of these cells was tested by inoculation into SCID mice. After 105 days after injection, mammary tumor developed from the BP1E cell line (Figure 4). None of the other BP clones or those derived from DMBA or MNNG-treated cells formed tumors (Table 2).

Figure 4. Diagrammatic representation of the evolution of MCF-10F cells treated with the chemical carcinogens.

Table 2. Expression of transformed phenotypes in MCF-10F and its derivatives1

Phenotypes

#130

MCF-10F

BP1

BP1E

In vitro Growth

  • Doubling time (hr)

N/A

93

80

44

Anchorage-Independent Growth

  • Survival Efficiency (%)

N/A

91.9 ± 35.0

N/A

N/A

  • Colony Efficiency (%)

N/A

0

45 ± 12

88 ± 10

  • Day 7 Colony Size (mm2)

N/A

0

167 ± 26

687 ± 28

Duct-like Formation in Collagen Gel

N/A

+

+/-

-

Invasiveness Index in Matrigel

N/A

130 ± 7

457 ± 43

820 ± 181

Tumorigenesis in SCID Mice

  • No. Cells Injected (x 106)

N/A

7 - 30

20

10

  • No. Animals w/ Tumors/Total No.

N/A

0/5

0/8

13/13

  • Average Tumor Size (mm3)

N/A

0

0

9.5

  • Latency Period (days)

N/A

?

?

101

Notes: 1. This table is modified from ref. 38.

These results led us to conclude that chemical carcinogens induce the expression of the definitive neoplastic phenotype, tumorigenesis, as long as HBEC are immortalized prior to carcinogen exposure. This model allowed us to isolate clone of cells expressing different stages of progression to neoplastic transformation, which will certainly facilitate our studies on the molecular mechanisms of cell transformation. Specifically, our in vitro model will allow us to answer the following important questions: 1. Which is the molecular mechanism of cell immortalization? 2. Which are the genetic changes involved in the process of cell transformation? 3. Which are the genetic changes related to the process of tumorigenesis? 4. Which of the genes identified in the process of tumorigenesis are important to revert the process? and 5. Which of the genetic changes are relevant to the process of neoplastic development in vivo?

5. MOLECULAR MECHANISMS OF CELL IMMORTALIZATION

5.1. Activation of telomerase

There is evidence that the repetitive TTAGGG sequences located at the ends of human chromosomes (i.e., telomeres) may act as a molecular mitotic clock (39). It is generally believed that each successive genome replication is accompanied by gradual shortening of 50-200 bp due to incomplete replication of the 3’ ends and cellular senescence occurs when telomeres reach a critically-short length that replication of the genome can not be maintained (22). Stabilization of the telomeric sequences at the ends of chromosomes, which is required for the continuous proliferation of immortal cells, involves the activation of the enzyme telomerase, which adds TTAGGG repeats to the 3’ ends of chromosomes (40, 41). The genetic nature of cellular senescence implicates repression of telomerase as a key element of cell immortalization (41). Elevated levels of telomerase activities have been detected in a number of immortal cell lines and human tumor tissues (42, 43). Our observation indicates that cell telomerase is expressed in immortal MCF-10F cells but not in the mortal #130 cells, and telomere lengths that have become shorter in the #130 cells, have been maintained with no further shortening due to telomerase activation in the immortal MCF-10F cells (44), suggesting that telomerase reactivation may be one of the mechanisms leading to the spontaneous immortalization of MCF-10F cells. In addition, we have evidence that telomerase activity may be regulated by the Ca++ concentration in the medium (44).

5.2. Abrogation of cell cycle control

Result from cell fusion studies indicates that the phenotype of cellular senescence is dominant and immortality results from recessive changes in normal regulatory genes (45). Conceivably, inactivation of the genes that restrict cell cycle progression is essential to cell immortalization. Cyclin-dependent kinase (CDK) complexes and their inhibitors are essential components of cell cycle machinery, controlling cell cycle arrest in the G1 phase of the cell cycle. Since p53 acts to regulate cell cycle progression through transcriptional activation of p21WAF-1, an inhibitor of all G1 CDKs (46), abrogation of p53 function has been implicated in the immortalization of HBEC. Insertional mutation at codon 247 of the p53 gene has been implicated in spontaneous immortalization of MCF-10F cells (47). Spontaneous immortalization in vitro has been observed in HBEC from a Li-Fraumeni patient with a point mutation in the p53 gene (41) and introduction of a single-amino acid deletion mutant (del239) of p53 gene abrogates wild-type p53-mediated cellular responses and induces immortalization of HBEC (48). A recent study indicates that alterations in p53 appear to be important in overcoming the M1 blockade (49). However, introduction of seven missense mutants of p53 genes failed to induce immortalization in the same cell line (48), even though all of these p53 mutants have been shown to abrogate p53-mediated transactivation in other cell types (50). In addition, the immortalized MCF-10F cells are still able to produce the wild-type p53 protein (7) and maintain wild-type p53-mediated functional responses, such as expression of p21WAF-1 (51) and mdm2 (7). Therefore, the role of p53 in immortalization of HBEC needs further evaluation.

The CDK-4 inhibitor (CDKN2), commonly referred to as p16, is also an inhibitor of the cell cycle and has been localized to 9p21-22 (52). Homozygous deletion of this chromosomal subregion has been observed in the immortalized MCF-10F cells (53), which contain a balanced reciprocal translocation, t(3;9) (3p13;9p22) (ref 12). Similarly, loss of the 9p21 subregion has been correlated with the acquisition of an immortal phenotype of neoplastic human head and neck keratinocyte cell lines (54). Clearly, these results suggest a potential role of CDK2 in the control of immortalization of human breast epithelial cells.

Another inhibitor of the cell cycle, prohibitin, has been implicated in the process of cell immortalization (55, 56). Prohibitin gene is localized to chromosome 17q21 (57) where mutations have been reported in certain forms of breast cancer (58), suggesting that it may be a tumor suppressor gene. Lack of heterozygosity has been documented in immortalized cell lines (55). Expression of prohibitin gene produces a 30kD-protein that inhibits cell cycle transition and DNA synthesis in normal cells (59). The 3’ untranslated region of prohibitin gene has been shown to function as a trans-acting regulatory RNA (i.e. riboregulator) crucial to its antiproliferative activity (56).

5.3. Genes preferentially expressed during cell immortalization

As further efforts to identify genes underlying the process of immortalization, we have performed subtractive hybridization and differential display analysis between immortal MCF-10F and its parental mortal #130 cells. Using a 10F(+)/130(-) subtractive cDNA library, we isolated more than 15 clones. Analysis of these clones showed that one of these clones contains sequences identical to H-ferritin (Figure 5) (60). Up-regulation of H-ferritin may be a source of iron necessary for growth and clonal expansion. Ferritin iron, once released, may increase the level of reactive iron, leading to an increase in oxygen free radical generation, oxidative DNA damage and mutation. Amplification of H-ferritin gene (61) and overexpression of H-ferritin protein (62) have been associated with the progression of human breast cancer. The role of H-ferritin in the immortalization of human breast epithelial cells is unclear and will be fully evaluated by gene transfection studies.

Figure 5. Differential expression of mRNA of H-ferritin in the transformed HBEC (Reproduced with permission from Ref. 60).

Recently, we have observed an increase in the expression of a cDNA, namely, calcium binding protein (CaBP), in the immortalized cells by differential display analysis (Figure 6) (63). Sequence analysis revealed that the CaBP cDNA is S100P Ca++-binding protein. Since Ca++ plays an important role in the spontaneous immortalization of MCF-10F human breast epithelial cells (13), it is conceivable that an increase in the expression of the S100P Ca++-binding protein may facilitate the process of cell immortalization. Further characterization of other cDNA clones identified by differential analysis is still in progress.

Figure 6. Northern blot hybridization using total RNA from immortal (MCF-10F), mortal (#130) and primary (#244) human breast epithelial cells showing a 0.6 kb transcript from the isolated calcium-binding protein gene (10F-D or S100P). The gene is expressed in the immortalized cells but not in the mortal or primary cells.

6. MOLECULAR MECHANISMS OF CELL TRANSFORMATION

6.1. Epigenetic mechanisms

The support for epigenetic mechanisms mainly stems from the fact that the efficiency of cell transformation induced by chemical carcinogens far exceeds the rate of genomic mutations imposed by the treatment (64). This notion appears to receive further support from our observations that the expression of transformed phenotypes, such as increased colony efficiency in agar methocel, was maximal shortly after exposure to chemical carcinogens and declined gradually during subsequent passage of the treated cells (Figures 7 & 8). However, the possibility remains that the cells transformed via the genetic mechanisms were gradually overwhelmed by the surrounding non-transformed cells during prolonged culture. This possibility should be examined in a suitable system in the future.

Figure 7. Survival efficiency of MCF-10F cells treated with the chemical carcinogens. MCF-10F cells were treated once (panel A) or twice (panel B) in a 7-day period. The treated cells were plated in agar methocel at every 2 passages after the treatment and allowed to grow anchorage-independently for 21 days.

Figure 8. Colony efficiency of MCF-10F cells treated with the chemical carcinogens. MCF-10F cells were treated once (panel A) or twice (panel B) in a 7-day period. The treated cells were plated in agar methocel at every 2 passages after the treatment and allowed to grow anchorage-independently for 21 days.

6.2. Genetic mechanisms

While epigenetic mechanisms of cell transformation represent a valid alternative, genetic alterations are generally perceived as the cornerstone of neoplastic development. Conceivably, neoplastic transformation occurs as a consequence of cumulative genetic alterations in regulatory mechanisms influencing cellular proliferation and/or programmed cell death or apoptosis. To delineate the molecular mechanisms responsible for cell transformation, we studied the effects of the chemical carcinogens on the expression of bcl-2, an apoptosis inhibitor (65) that is highly expressed in breast carcinomas with a low apoptotic index (66, 67), and cyclin D1, a proliferation-associated gene which is frequently amplified or overexpressed in all forms of breast carcinoma (68-72). Our results indicated that the levels of bcl-2 and cyclin D1 expression were unaffected by chemical carcinogen treatments during the initial phases of cellular transformation in vitro (14). Therefore, the role of bcl-2 and cyclin D1 in the etiology of human breast cancer, if any, appears to be subsequent to the initial stage of neoplastic transformation. The roles of other oncogenes and tumor suppressor genes in chemical carcinogenesis need to be evaluated.

7. GENOMIC CHANGES IN THE IMMORTALIZATION AND TRANSFORMATION OF HBEC

7.1. Genomic changes in cell immortalization

Senescence of human fibroblasts has been associated with genetic determinant(s) on chromosome 4 (73) and the long (q) arm of chromosome 1 (74). Karyotyping analyses of 5 spontaneously-immortalized human breast epithelial cell lines have identified several common chromosomal alterations including loss of chromosome 20p and gain of chromosome 1q (75). The most common genomic change in SV40-immortalized human cells is the loss of chromosome 6 (76). Recently, alterations at several other chromosomal loci (e.g. 20q13.2, 6q26-27) have been implicated in immortalization of various epithelial cells with viral oncogenes (77, 78). However, the nature and the function of genes located in these chromosomal loci remains to be defined. In our laboratory, we have determined that the immortalization of the MCF-10F is associated with the mutation at exon 7 of the TP53 locus (47) (Figure 9), supporting the notion that inactivation or loss of function-mutation of the p53 gene is critical in the early stages of breast cancer progression (79, 80). In addition to further evaluation on the role of p53 in immortalization of HBEC, our laboratory is currently pursuing studies to determine further genomic changes such as microsatellite instability (MSI) and its underlying mechanisms that may play a role in the immortalization of HBEC.

Figure 9. Figure 9 A-E. Direct DNA sequencing of the PCR amplified products generated from exon 7 of the p53 gene of A: placenta; B: MCF-10M cells; C: MCF-10F cells; D: D3-1 cells; and E: MCF-7 cells. An insertional mutation of a T at codon 254 was observed in MCF-10F (C) and its derivative D3-1 (D) cells, causing a frame shift following codon 254. Right-hand panel: PCR-SSCP analysis of exons 4-9 of the p53 gene. Exons 4, 5, 6 and 8-9 showed identical appearance in placenta and MCF-10M. A conformational shift in exon 7 was observed in three MCF-10F cell lines tested and the same shift was maintained in MCF-10F-derived transformed cell lines (BP1, BP1E, D3 and D3-1) (Reproduced with permission from Ref. 47).

7.2. Genomic changes in cell transformation

Genomic alterations have been recognized as a hallmark of cancer progression (81-85). The unstable changes of microsatellite, or very short simple repetitive sequences, designated as (CA)n, that are distributed throughout the genome (86, 87) represent such alterations. Its association with human malignancies has been extended from colorectal cancer (88-90) to neoplasms of the neck and head (91), lung (92), skeletal muscle (93), lympho-hematologic system (94), skin such as melanoma (95), prostate (96), gastrointestinal system (97), urinary bladder (98), liver (99), neurologic system (100, 101), cervix (102), endometrium (103), as well as breast (104-112). These findings indicate that MSI is associated with the general process of carcinogenesis.

MSI has been associated with the progression of breast cancer. However, its exact timing is controversial and its specific functional roles are not clear. It may be present as an early (105-108, 112), or a late event (113), or both (114), or not correlated (115, 116) during the breast carcinogenesis, dependent upon the markers (locus specific for each chromosome) used, and samples tested. This assumption is supported by the study of Aldaz et al (114) showing that some chromosomal loci might be involved early, while others late during the progression of human breast cancers. Therefore, the elucidation of this genomic phenomenon can be further clarified by analysis with a comprehensive array of markers in an in vitro system such as ours that is free of affecting factors from variations among individuals and yet consists of various stages of cell transformation. More importantly, such studies may lead to the determination of underlying mechanisms such as defects of DNA mismatch repair genes that have been documented in colorectal, endometrial, ovarian, and prostate cancers (reviews in 117, 118), which have emerged as another type of factors as important as the tumor suppressor genes and oncogenes in breast carcinogenesis. Specifically, several questions exist: 1. Does MSI occur in preference to a particular chromosome(s) at a specific locus? 2. Is this instability really correlated with the phenotypic progression of human breast cancer and thus does occur in a sequential order? 3. What are the specific underlying mechanisms, such as mismatch repair gene defects, or DNA replication errors, or others?

We have pursued the first question by analyzing a total of 466 microsatellite loci on all the chromosomes in transformed HBECs representing the early and intermediate stages of cell transformation (119). These markers were selected to represent 38-96% the banded regions (according to the Human Genome Maps V) (120), taking into consideration of locations where tumor suppressor genes, oncogenes, DNA repair genes, and other cancer or cell growth regulation-associated genes are documented or postulated to be situated. Interestingly, we were able to detect MSI in only a very small number of loci; 0.64% (3/466), or 0.43% (2/466) of the markers analyzed were found in the BP-, or DMBA-transformed HBECs, respectively. These changes were exclusively found in the chromosomal regions of 11q25 at locus D11S912 and 13q12-13 at loci D13S260 and D13S289 in the BP-cells, or the 13q12-13 region at loci D13S260 and 16q12.1 at D13S260 and 16q12.1 at D16S285 in the DMBA-cells (119, 121) (Figure 10). Furthermore, the occurrence of MSI among these loci in the BP-cells seems to reflect a sequential order; i.e., 11q25 (D11S912) in the BP1 cells, followed by 13q12-13 (D13S260) and then another locus of 13q12-13 (D13S289) in the BP1E cells. However, this tendency is not seen in the DMBA cells. Our data have provided direct evidence that MSI is associated with the early and intermediate transformation of HBEC, during which only a very small proportion of loci are affected, and that the involvement of these loci on chromosomes 11 and 13 may be correlated with the progression of HBEC transformation in vitro. This finding supports that carcinogen-transformed HBECs, and presumably sporadic breast carcinomas, are also characterized with a mutator phenotype (122), that appears early as a driving force in tumor progression (review in 84, 123).

Figure 10. Microsatellite instability in the transformed HBECs (1: MCF-10F; 2: BP1; 3: BP1E) and human breast lesions (N: Normal; D: DHP; C: CIS; I: INV).

The carcinogenic mechanisms of benzo(a)pyrene are postulated to be related to its ability to produce G->T mutations, which affect nucleotides in GC-rich stretches of DNA (124), as showed by mutations of p53 at codons 154, 173, 248, 266, 173 and 277 (125, 126). We hypothesize that BP may cause additional mutations in TP53 and other target genes in the transformed cells, which hence contribute to the genomic instability detected in these cells. This is supported by the evidence that germline mutations of p53 are associated with the genomic instability with Li-Fraumeni syndrome (review in 127). Other mechanisms that may contribute to genomic instability include defects in DNA mismatch repair genes (reviews in 117, 118), DNA replication error and DNA repair defects (reviews in 84, 128, 129).

8. GENOMIC CHANGES IN HUMAN BREAST LESIONS

In order to test whether similar changes of microsatellits are present in human breast carcinogenesis in vivo, we have also done microsatellite polymorphism analysis in genomic DNA extracted from breast lesions of three different categories: ductal hyperplasia (DHP), carcinoma in situ (CIS), and invasive carcinoma (INV). Using the array of 7 markers including D11S912, D11S940, D13S260, D13S289, D13S267, D16S285 and D17S855, we analyzed samples from 21 patients (Table 3) (119). Among these markers, D11S912 showed MSI in 10/19 (53%) of all samples including 2/8 of the preneoplastic lesion DHP, 8/15 of CIS, and 3/5 of INV (Figure 10; Table 3), in agreement with its early appearance in the transformed HBEC (i.e., BP1 cells) (see above). MSI of marker D13S260, which was absent in DHP, but present in 5/16 (31%) and 3/4 (75%) of CIS and INV, respectively (Figure 10; Table 3), suggesting a correlation with the progression and also a consistence with its alteration in the transformed HBEC (i.e., BP1E cells). In addition, the high MSI incidence in all samples for markers D11S912 (53%), D13S260 (23%), D13S289 (36%) and D13S267 (45%), as compared to lower rates of D11S940 (10%), D16S285 (6%) and D17S855 (9%) (Table 3) may suggest that instability may prefer to occur in these loci during breast carcinogenesis, also in good agreement with the data from the transformed HBEC system (except for D13S267).

Table 3. Microsatellite instability in human breast lesions

MARKER

Type

TOTAL

%

DHP*

%

CIS

%

INV

%

D11S912

di

10/19

53

2/8

25

8/15

53

3/5

33

D11S940

di

2/20

10

0/9

0

1/16

6

1/5

20

D13S289

di

4/11

36

0/5

0

2/7

29

2/2

100

D13S260

di

5/19

23

0/7

0

5/16

31

3/4

75

D13S267

di

9/20

45

1/7

14

9/16

56

2/4

50

D16S285

di

1/16

6

0/4

0

1/15

7

0/3

0

D17S855

di

1/11

9

0/4

0

0/8

0

1/4

25

Notes: *DHP: ductal hyperplasia; CIS: carcinoma in situ; INV: invasive carcinoma; di: dinucleotide repeats.

9. FUNCTIONAL ROLES OF CHROMOSOMES 11 AND 17 IN THE EXPRESSION OF TRANSFORMED PHENOTYPES OF HBEC

The functional role of specific genes on a chromosome can be determined by microcell fusion technique (130, 131), in which a monochromosome is introduced into the target cells. For example, tumor suppressor genes can exert a reversion of tumorigenic or transformed phenotypes that can be demonstrated as a reduction in or a total loss of the neoplastic/transformed phenotypes, together with at various degree the reappearance of normal phenotype, as a result of the inhibition of activities of specific oncogenes or the replacement of intact tumor suppressor genes (132).

The application of microcell-mediated chromosome transfer (MMCT) technique (130) has led to the determination of genes on a candidate chromosome responsible for genetic disorders or neoplastic phenotypes (133-135). For example, growth/tumor suppressor activities have been detected in chromosomes 1, 3, 5, 6, 7, 9, 11, 17 and X in various human tumor cell lines (135-154). Senescence genes have been found on chromosomes 1, 2, 3, 4, 6, 7, 10, 11, 18 and X (for reviews, see 155). Putative tumor suppressor genes have been mapped to chromosomes 1q23-qter (156), 1 (157), 3p (153, 158, 159), 6q21-q23 and/or 6q26-q27 (147, 160, 161), 11 (160-162), 17 (163) and 17q (151, 154). While putative DNA repair genes have been localized to chromosomes 2 (164, 165), 3 (166), 8 (167) and 11 (168, 169), putative metastasis genes are found on chromosomes 8p23-q12 (170, 171), 10q (170), 11p13-p11.2 (170), 11 (172) and 17pter-q23 (173). Together, the application of this technique has allowed for a functional testing of genetic material residing in a specific chromosome that promises further characterization of specific genes affecting the phenotypes of a tumor cell.

As discussed above, genetic alterations in specific genes such as mutations of TP53 and c-H-ras genes, amplification and/or overexpression of c-erbB, c-myc and MDM2 genes, and instability of microsatellites in certain regions of chromosomes are associated with the phenotypic progression of HBEC during transformation (Figure 11). However, it is not clear whether any of these changes play a functional role in the ultimate expression of the transformed phenotypes of these HBECs.

Figure 11. Genomic alterations in the evolution of immortalization and transformation of HBECs.

In order to obtain such information, we have introduced normal human fibroblast-derived chromosome 11, or 17, respectively, into the transformed BP1E cells, through MMCT technique (174). We have found that BP1E cells transferred with chromosome 11 or 17, designated as BP1E-11neo, and BP1E-17neo cells, showed significant morphologic regression such as growth inhibition (e.g., contact inhibition, slow or no division) and characteristic of senescence in most or all clones, which lost the ability to divide and eventually died during a selection incubation period up to 6-12 months. Fluorescence in situ hybridization (FISH) analysis of expandable clones showed that a donor chromosome 11 was present in the BP1E-11neo cells, and that a donor chromosome 17 was present in the BP1E-17neo cells. Functional analyses showed that the BP1E-11neo cells exhibited a reduction in growth rate by 50% and in colony efficiency in agar-methocel by 55% (Table 4). Similarly, the BP1E-17neo cells lost its growth rate by 90% and its capacity to form colonies in agar-methocel completely, while regained the ability to form ductule-like structure in collagen-matrix and became intolerant to high calcium concentration in the medium (1.04 mM) (Table 4). These data indicate that both chromosomes 11 and 17 may play a functional role in the expression of transformed phenotypes and that they may harbor tumor suppressor genes or senescence genes, whose normal functions/regulations may have been disrupted in the BP1E cells. Further studies should be directed towards the mapping and cloning of specific genes on these chromosomes and their roles in the progression of transformed phenotypes.

Table 4. Analysis of BP1E-11neo and BP1E-17neo Cells

Cells

G-418

Resistance1

pSV2neo

Sequence2

Extra (Donor)

Chromosome3

Growth Properties

Rate4

Anch. independence5

Ductulogenesis6

Ca++ Resistance7

BP1E control

No

No

No

100%

100%

-

Yes

BP1E-11neo

Yes

Yes

Yes

50%

42%

N/A

Yes

BP1E-17neo

Yes

Yes

Yes

10%

0%

+

No

Notes: 1. G-418 resistance: The BP1E cells could not survive a concentration of 100 mg/ml within a week; while the BP1E-11neo and BP1E-11neo cells were selected and routinely maintained in 400 mg/ml in the media.

2. pSV2neo sequence: The donor chromosomes 11 or 17 are tagged with vector pSV2neo. A partial sequence of 466 basepairs (GenBank Access number: U02434) of this vector was amplificed from the genomic DNA by PCR, transferred to nylon membrane, and hybridized with the pSV2neo probe to confirm the specificity of PCR products.

3. Extra (donor) chromosome: The detection of the donor chromosome 11 or 17 in the microcell hybrids BP1E-11neo, or BP1E-17neo was done by fluorescence in situ hybridization (FISH) using digoxigenin-labeled chromosome 11 or 17 coatsome (painting) probe (Oncor), together with a biotin-labeled pSV2neo probe. The presence of the donor chromosome 11 or 17 was judged by the co-presence of its distinct morphology in the metaphase from the host chromosome and the biotin signals on it.

4. Growth rate: It was determined by the total cell count at the end of a five-day growth on 24-well chambers (Costar). The growth rate of the control BP1E cells has been arbitarily set as 100%, and the tested as proportions of the control.

5. Anch. independence (anchorage independent growth): Colony formation of the cells were tested in 0.8% methocel semi solid gel on agar base for 21 days. The colony efficiency of the control has been arbitarily set as 100%, and the tested as a ratio of the control. The average colony size between the BP1E control and the BP1E-11neo cells was not different.

6. Dutulogenesis: This parameter was evaluated by growing cells in collagen matrix for 21 days. Although BP1E cells remained growing in cell clusters, the BP1E-17neo cells were able to regain the ability to form ductule-like structure in the matriz. "N/A" indicates data "not available."

7. Ca++ Resistance: The BP1E cells have adapted to grow in the high calcium contration (1.05 mM) in the medium. The introduction of chromosome 11 did not affect this growth property of the cells. However, the same calcium concentration in the medium induced senesent phenotypes of the BP1E-17neo cells, which is significantly reversed by replacing with low calcium concentration (0.04 mM) in the medium.

10. SUMMARY AND PERSPECTIVES

Human breast cancer remains the most common malignancy and second leading cause of cancer death in the American women. The ultimate cure of this disease relies on a better understanding of the mechanisms underlying the initiation and progression of this disease. The neoplastic transformation of HBEC in vitro represents a successful model for obtaining knowledge on the molecular and biological alterations that may contribute to the tumorigenic mechanisms (reviews in 4, 7, 20, 175, 176). We have presented here a current understanding of chemically transformed HBEC in the following aspects: 1. Factors affecting the transformation of HBEC such as genetic predisposition and differentiation status and prior immortalization; 2. New targets for studying the mechanism of cell immortalization such as alterations in telomerase activity and differential expression of cell cycle dependent genes as well as others recently isolated through differential cloning such as H-ferritin, and a calcium binding protein; 3. Epigenetic and genetic mechanisms underlying cell transformation; 4. The association of microsatellite instability in specific loci on chromosomes 11, 13, and 16 with the progression of cell transformation; and 5. The application of microcell mediated chromosome transfer technique as an approach to testing the functional role of specific genes whose dysregulation or loss of function may contribute to the ultimate cell transformation. Further efforts in this cell system will be directed to determine the roles of identified molecular changes as well as the mapping/cloning of tumor suppressor or senescence genes such as those that may reside on chromosome 11 or 17.

11. ACKNOWLEDGMENTS

This work was supported by Grants CA64896 and CA67238 awarded by National Cancer Institute, and Grant ES07280 awarded by the National Institute of Environmental Health Sciences, USA. 2Fellow, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil. Abdel Zekri is a visiting scientist, Cairo University, Cairo, Egypt. Xiaoqi Yang, Yun-Fu Hu contributed equally to this work.

12. REFERENCES

1. S.L. Parker, T. Tong, S. Bolden & P.A. Wingo: Cancer statistics 1996. CA Cancer J Clin 46, 5-27 (1996)

2. S.L. Parker, T. Tong, S. Bolden & P.A. Wingo: Cancer Statistics. CA Cancer J Clin 47, 5-27 (1997)

3. J.W. Simons: Genetic, epigenetic, dysgenetic, and non-genetic mechanisms in tumorigenesis. Crit Rev Oncogenesis 6, 261-73 (1995)

4. R. Holliday: Neoplastic transformation: the contrasting stability of human and mouse cells. Cancer Survey 28, 103-15 (1996)

5. D. B. Couch: Carcinogenesis: basic principles. Drug Chem Toxicol 19, 133-48 (1996)

6. J. Russo, G. Calaf & I.H. Russo: A critical approach to the malignant transformation of human breast epithelial cells with chemical carcinogens. Crit Rev Oncogenesis 44, 403-17 (1993)

7. J. Russo, N. Barnabas, N. Higgy, A.M. Salicioni, Y.L. Wu & I.H. Russo: Molecular basis of human breast epithelial cell transformation. In Breast Cancer. Advances in biology and therapeutics. Eds: Calvo F, Crepin M and Magdelenat H, John Libbey Eurotext (1996) pp33-43.

8. E. Farber: The multistep nature of cancer development.

Cancer Res 44, 4217-23 (1984)

9. G. Klein & E. Klein: Evolution of tumors and the impact of molecular oncology. Nature 315, 190-5 (1985)

10. D.J. Page & W.D. Dupont: Anatomic markers of human premalignancy and risk of breast cancer. Cancer 66, 1326-35 (1990)

11. J. Russo, D. Reina, J. Frederick & I.H.Russo: Expression of phenotypical changes by human breast epithelial cells treated with carcinogens in vitro. Cancer Res 48, 2837-57 (1988)

12. H.D. Soule, T.M. Maloney, S.R. Wolman, W.D. Peterson, R. Brenz, C.M. McGrath, J. Russo, R.J. Pauley, R.F. Jones & S.C.Brooks: Isolation and characterization of a spontaneously immortalized human breast epithelial cell line. MCF-10. Cancer Res 50, 6075-86 (1990)

13. J. Russo, G. Calaf, N. Sohi, Q. Tahin, P.L. Zhang, M.E. Alvarado, S. Estrada & I.H. Russo: Critical steps in breast carcinogenesis. Ann NY Acad Sci USA 698, 1-20 (1993)

14. Y.F. Hu, I.H. Russo, U. Zalipsky, H.T. Lynch & J. Russo: Environmental chemical carcinogens induce transformation of breast epithelial cells from women with familial history of breast cancer. In Vitro Cell Dev Biol 33, 495-98 (1997)

15. R.G. Ham: Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc Natl Acad Sci USA 53, 288-93 (1965)

16. S.L. Hammond, R.G. Ham & M.R. Stampfer: Serum-free growth of human mammary epithelial cells. rapid clonal growth in defined medium and extended serial passage with pituitary extract. Proc Natl Acad Sci USA 81, 5435-9 (1984)

17. L. Tait, H.D. Soule & J. Russo: Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10. Cancer Res 50, 6087-6094 (1990)

18. M.J. Mills, M.J. Mousalli & J. Russo: DNA-labeling index and in vitro growth of mammary epithelium are modulated by the stage of gland development. Proc Int Assoc Breast Cancer Res, Miami, FL (1987)

19. M.J. Mousalli, M.J. Mills & J. Russo: Influence of mammary gland development on its epithelium's growth potential in tissue culture. Proc Int Assoc Breast Cancer Res, Miami, FL (1987)

20. V. Band: Preneoplastic transformation of human mammary epithelial cells. Sem Cancer Biol 6, 185-92 (1995)

21. A. Derventzi, S.I.S. Rattan & E. Gonos: Molecular links between cellular mortality and immortality. Anticancer Res 16, 2901-10 (1996)

22. U. Hopfer, J.W. Jacobberger, D.C. Gruenert, R.L. Eckert, R.S. Jat & J.A. Whitsett: Immortalization of epithelial cells. Am J Physiol 270, c1-c11 (1996)

23. R.F. Newbold: Multistep malignant transformation of mammalian cells by carcinogens: Induction of immortality as a key event. In: Carcinogenesis Vol 9. Eds: Barrett JC, Tennant RW, Raven Press, NY (1985) pp17-28.

24. V. Band, D. Zagetowski, V. Kulesa & R. Sager: Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements. Proc Natl Acad Sci USA 87, 463-7 (1990)

25. V. Band, S. Dalal, L. Delmolino & E.L. Androphy: Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells. EMBO J 12, 1847-52 (1993)

26. D.E. Wazer, X.L. Liu, Q. Chu, Q. Gao, V. Band: Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7. Proc Natl Acad Sci USA 92, 3687-91 (1995)

27. A. Yilmaz, A.C. Gaide, B. Sordat, Z. Borbenyi, H. Lahm, A. Imam, M. Schreyer & Odartchenko: Malignant progression of SV40-immortalized human milk epithelial cells. Br J Cancer 68, 868-73 (1993)

28. P. Berthon, G. Goubin, B. Dutrillaux, A. Degeorges, A. Faille, C. Gespach & F. Calvo: Single step transformation of human breast epithelial cells by SV40 large T oncogene. Int J Cancer 52, 97-7 (1992)

29. J. Russo & I.H. Russo: The etiopathogenesis of breast cancer prevention. Cancer Letters 90, 81-89 (1995)

30. Y.F Hu, I.H. Russo, X. Ao & J. Russo: Mammary-derived growth inhibitor (MDGI) cloned from human breast epithelial cells is expressed in fully differentiated lobular structures. Int J Oncol 11, 5-11 (1997)

31. C.M. Phelan, C. Larsson, S. Baied, P.A. Futreal, M.H. Ruttledge, K. Morgan, P. Tonin, H. Hung, R.G. Korneluk, M.N. Pollak & S.A. Narod: The human mammary-derived growth inhibitor (MDGI) gene: Genomic structure and mutation analysis in human breast tumors. Genomics 34, 63-8 (1996)

32. M. Genardi, H. Tsihira, D.E. Anderson & G.F. Saunders: Distal deletion of chromosome 1p in ductal carcinoma of the breast. Am J Hum Genet 45, 73-82 (1989)

33. H.T. Huynh, C. Larsson, S. Narod & M. Pollak: Tumor suppressor activity of the gene encoding mammary-derived growth inhibitor. Cancer Res 55, 2225-31 (1996)

34. K.A. Traul, K. Takayama, V. Kachevsky, R. J. Hink & J.S. Wolff: A rapid in vitro assay for carcinogenicity of chemical substances in mammalian cells utilizing an attachment independence endpoint. 2. Assay validations. J Appl Toxicol 1, 190-5 (1981)

35. D.L. Putnam, D.K. Park, J.S. Rhim, A.F. Stever & R.C. Ting: Correlation of cellular aggregation of transformed cells and their growth in soft agar and tumorigenic potential. Proc Soc Exp Biol Med 155, 487-94 (1977)

36. H.S. Feigelson, R.R. Ross, M.C. Yu, G.A. Coetzee, K.V. Reichardt & B.E. Henderson: Genetic susceptibility to cancer from exogenous and endogenous exposures. J Cell Biochem 25S, 15-22 (1996)

37. M.N. Gould, R.A. Lubet, G.J. Kellof & J.D. Haag: Inherited susceptibility and acquired allelic imbalance in rat mammary carcinogenesis. J Cell Biochem 25S, 37-40 (1996)

38. G. Calaf & J. Russo: Transformation of human breast epithelial cells by chemical carcinogens. Carcinogenesis 14, 483-92 (1993)

39. C.B. Harley: Telomere loss: mitotic clock or genetic time bomb? Mutation Res 256, 271-82 (1991)

40. E.H. Blackburn: Telomerase. Annu Rew Biochem 61, 113-29 (1992)

41. J.W. Shay, W.E. Wright & H. Werbin: Toward a molecular understanding of human breast cancer: A hypothesis. Breast Cancer Res Treat 25, 83-94 (1993)

42. S. Bacchetti & C.M. Counter: Telomeres and telomerase in human cancer. Int J Oncol 7, 423-32 (1995)

43. A.A. Avilion, M.A. Piatyszek, J. Gupta, J.W. Shay, S. Bacchetti & C.W. Greder: Human telomerase RNA and telomerase activity in immortal cells lines and tumor tissues. Cancer Res 56, 645-50 (1996)

44. Y.F. Hu, I.H. Russo, C.M. Slater & J. Russo: Regulation of telomerase activity by extracellular calcium in human breast epithelial cells (manuscript in preparation).

45. O.M. Pereira-Smith & J.R. Smith: Evidence for the recessive nature of cellular immortality. Science 221, 964-6 (1983)

46. Y. Xiong, G.J. Hannon, D. Zhang, D. Casso, R. Kobayashi & D. Beach: p21 is a universal inhibitor of cyclin kinases. Nature 366, 701-4 (1993)

47. N. Barnabas, R. Moraes, G. Calaf, S. Estrada & J. Russo: Role of p53 in MCF-10F cell immortalization and chemically-induced neoplastic transformation. Int J Oncol 7, 1289-96 (1995)

48. Q. Gao Q, S.H. Hauser, X.L. Liu, D.E. Wazer, H. Madoc-Jones & V. Band: Mutant p53-induced immortalization of primary human mammary epithelial cells. Cancer Res 56, 3129-33 (1996)

49. L.S. Gollahon & J.W. Shay: Immortalization of human mammary epithelial cells transfected with mutant p53 (273his). Oncogene 12, 715-25 (1996)

50. J.Y. Chen, W.D. Funk, W.E. Wright, J.W. Shay & J.D. Minna: Heterogeneity of transcriptional activity of mutant p53 proteins and p53 DNA target sequences. Oncogene 8, 2159-66 (1993)

51. Y.F. Hu & J. Russo: Expression of p21WAF-1 in MCF-10F cells. (unpublished data).

52. A. Kamb, N.A. Gruis, J. Weaver-Feldhaus, Q. Liu, K. Harshman, S.V. Tavtigian, E. Stockert, R.S. Day, B.E. Johnson & M.H. Skolnick: A cell cycle regulator potentially involved in genesis of many tumor types. Science 264, 436-40 (1994)

53. A.J. Brenner & C.M. Aldaz: Chromosome 9p allelic loss and p16/CDKN2 in breast cancer and evidence of p16 inactivation in immortal breast epithelial cells. Cancer Res 55, 2892-95 (1995)

54. O. Loughran, K.G. Edinogton, I.J. Berry, L.J. Clarke & E.K. Parkinson: Loss of heterozygosity of chromosome 9q21 is associated with the immortal phenotypes of neoplastic human head and neck keratinocytes. Cancer Res 54, 5045-49 (1994)

55. E.R. Jupe, X.T. Liu, J.L. Kiehlbauch, J.K. McClung & R.T. Dell’Orco: Prohibitin antiproliferative activity and lack of heterozygosity in immortalized cell lines. Exp Cell Res 218, 577-80 (1995)

56. E.R. Jupe, X.T. Liu, J.L. Kiehlbauch, J.K. McClung & R.T. Dell’Orco: The 3’ untranslated region of prohibitin and cellular immortalization. Exp Cell Res 224, 128-35 (1996)

57. J.J. White, D.H. Ledbetter, R.L. Eddy, Jr, T.B. Shows, D.A. Stewart, M.J. Nuell, V. Friedman, C.M. Wood, G.A. Owens, J.K. McClung, D.B. Danner & C.C. Morton: Genomics 11, 228-30 (1991)

58. T. Sato, H. Saito, J. Swensen, A. Olifant, C. Wood, D. Danner, T. Sakamoto, K. Takita, F. Kasumi, Y. Miki, M. Skolnick & Y. Nakamura: The human prohibitin gene, located on chromosome 17q21, is mutated in sporadic breast cancer. Cancer Res 52, 1643-46 (1992)

59. M.J. Nuell MJ, D.A. Stewart, L. Walker, V. Friedman, C.M. Wood, F.A. Owens, J.R. Smith, E.L. Schneider, R.T. Dell’Orco, C.K. Lumpkin, D.B. Danner & J.K. McClung: Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells. Mol Cell Biol 11, 1372-81 (1991)

60. N.A. Higgy, A.M. Salicioni, I.H. Russo, P.L. Zhang & J. Russo: Differential expression of human ferritin H chain gene in immortal human breast epithelial cells MCF-10F. Mol Carcinog 20, 332-339, 1997.

61. E. Abruzzese, P.N. Rao, M.J. Pettenati, M.J. Kute, S.V. Torti & F.M. Torti: The ferritin H gene is amplified in human breast cancer carrying an 11Q13 amplification. Proc Am Assoc Cancer Res 38, 415 (1997)

62. D.C. Yang, J.F. Head, F. Wang & R.L. Elliott: Transferrin receptor mRNA and ferritin H-chain mRNA expression are associated with prognostic indicators of breast cancer. Proc Am Assoc Cancer Res 38, 419 (1997)

63. I.D.C.G. Silva, Y.F. Hu, X. Ao, I.H. Russo, A.M. Salicioni, X. Yang & J. Russo: S100P Ca+2-binding protein overexpression is associated with immortalization and neoplastic transformation of human breast epithelial cells in vitro and tumor progression in vivo. Oncogene (submitted).

64. B.S. Cha, W.G. Thilly & H. Zarbl: N-Nitroso-N-methylurea-induced rat mammary tumors arise from cells with preexisting oncogenic Hras1 gene mutations. Proc Natl Acad Sci USA 91, 3749-53 (1994)

65. D. Hockenbery, G. Nunez, C. Milliman & S.J. Korsmeyer: Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 349, 334-6 (1990)

66. W.K. Chan, R. Poulsom, Q.L. Lu, K. Patel, W. Gregory, C.J. Fisher & A.M. Hanby: Bcl-2 expression in invasive mammary carcinoma: correlation with apoptosis, hormone receptors and p53 expression. J Pathol 169, 153A (1993)

67. J.M. Gee, J.F. Robertson, I.O. Ellis, P. Willsher, R.A. McClelland, H.B. Hoyle, S.R. Kyme, P. Finlay, R.W. Blamey & R.I. Nicholson: Immunocytochemical localization of BCL-2 protein in human breast cancers and its relationship to a series of prognostic markers and response to endocrine therapy Int J Cancer 59, 619-28 (1994)

68. M.F. Buckley, K.J.E. Sweeney, J.A. Hamilton, R.L. Sini, D.L. Manning, R.I. Nicholson, A. DeFazio, C.K.W. Watts, E.A. Musgrove & R.L. Sutherland: Expression and amplification of cyclin genes in human breast cancer. Oncogene 8, 2127-33 (1993)

69. J. Bartkova, J. Lukas, H. Muller, D. Lutzhoft, M. Strauss & J. Bartek: Cyclin D1 protein expression and function in human breast cancer. Int J Cancer 57, 353-61 (1994)

70. C. Gillet, V. Fantl, R. Smith, C. Fisher, J. Bartek, C. Dickson, D. Barnes & G. Peters: Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res 54, 1812-7 (1994)

71. S.Y. Zhang, J. Camano, F. Cooper, X. Guo & A. Klein-Szanto: Immuno-histochemistry of cyclin D1 in human breast cancer. Am J Clin Path 102: 695-8 (1994)

72. D. Weistat-Saslow, M.J. Merino, E.M. Richard, J.A. Lawrence, R.F. Bluth, K.D. Wittenbel, J.F. Simpson, D.L. Page & P.A. Steeg: Overexpression of cyclin D mRNA distinguish invasive and in situ breast carcinomas from non-malignant lesions. Nature Med 1, 1257-60 (1995)

73. Y. Ning, J.L. Webber, A.M. Killary, D.H. Ledbetter, J.R. Smith & O.M. Pereira-Smith: Genetic analysis of indefinite division of human cells: Evidence for a cell senescence-related gene(s) on chromosome 4. Proc Natl Acad Sci USA 88, 5635-9 (1991)

74. O. Suguwara, M. Oshimura, M. Koi, L.A. Annab & J.C. Barrett: Induction of cellular senescence in immortalized cells by human chromosome 1. Science 247, 707-10 (1990)

75. S.R. Wolman, A.N. Mohamed, G.H. Heppner & H.D. Soule: Chromosomal markers of immortalization in human breast epithelium. Genes Chrom Cancer 10, 59-65 (1994)

76. F.A. Ray & P.M. Kraemer: Frequent deletions in nine newly immortal human cell lines. Cancer Genet Cytogenet 59, 39-44 (1992)

77. S.S. Banga, S.H. Kim, K. Hubbard, T. Dasgupta, K.K. Jha, P. Patsalis, R. Hauptschein, B. Gamberi, R. Dalla-Favera, P. Krarmer & H.L. Ozer: SEN6, a locus for SV-40-mediated immortalization of human cells, maps to 6q26-27. Oncogene 14, 313-21 (1997)

78. E. Savelieva, C.D. Belair, M.A. Newton, S. DeVries, J.W. Gray, F. Waldman & C.A. Reznikoff: 20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papilloma virus 16 E7 transformed human uroepithelial cells. Oncogene 14, 551-60 (1997)

79. E.Y.-H.P. Lee: Tumor suppressor genes and their alterations in breast cancer. Sem Cancer Biol 6, 119-25 (1995)

80. L.A. Donehower: Effects of p53 mutation on tumor progression: recent insights from mouse tumor models. Biochim Biophy Acta 1242, 171-6 (1996)

81. W.B. Coleman & G.J. Tsongalis: Multiple mechanisms account for genomic instability and molecular mutation in neoplastic transformation. Clin Chem 41, 644-57 (1995)

82. I. Bieche & R. Lidereau: Genetic alterations in breast cancer. Gene Chrom Cancer 14, 227-51 (1995)

83. J.R. Eshleman & S.A. Markowitz: Microsatellite instability in inherited and sporadic neoplasms. Curr Opinion Oncol 7, 83-9 (1995)

84. L.A. Loeb: Many mutations in cancer. Cancer Surveys 28, 329-42 (1996).

85. G.H. Heppner & F.R. Miller: The cellular basis of tumor progression. Int Rev Cytol 77, 1-56 (1997)

86. J.L. Weber & P.E. May: Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44, 388-96 (1989)

87. M. Litt & J.A. Luty: A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am J Hum Genet 44, 397-401 (1989)

88. L.A. Aaltonen, P. Peltomaki, F.S. Leach, P. Sistonen, L. Pylkkanen, J.P. Mecklin, H. Jarvinen, S.M. Powell, J. Jen, S.R. Hamilton, G. Petersen, K.V. Kinzler, B. Vogelstein & A. Dela Chaplelle: Clues to the pathogenesis of familial colorectal cancer. Science 260, 812-16 (1993)

89. S.N. Thibodeau, G. Bren & D. Schaid: Microsatellite instability in cancer of the proximal colon. Science 260, 816-9 (1993)

90. Y. Ionov, M.A. Peinado, S. Malkhosyan, D. Shibata & M. Perucho: Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363, 558-61 (1993)

91. A.K. El-Naggar, K. Hurr, V. Huff, G.L. Clayman, M.A. Luna & J.G. Batsakis: Microsatellite instability in preinvasive and invasive head and neck squamous carcinoma. Am J Pathol 148, 2067-72 (1996)

92. J. Adachi, M. Shiseki, T. Okazaki, G. Ishimaru, M. Noguchi, S. Hirohashi & J. Yokota: Microsatellite instability in primary and metastatic lung carcinomas. Gene Chrom Cancer 14, 301-6 (1995)

93. M. Visser, J. Bras, C. Sijmons, P. Devilee, L.C. Wijnaendts, J.C. van der Linden, P.A. Voute & F. Baas: Microsatellite instability in childhood rhabdomyosarcoma is locus specific and correlates with fractional allelic loss. Proc Natl Acad Sci USA 93, 9172-6 (1996)

94. R.B. Gartenhaus: Microsatellite instability in hematologic malignancies. Leukemia & Lymphoma 25, 455-61 (1997)

95. K. Peris, G. Keller, S. Chimenti, A. Amantea, H. Kerl & H. Hofler: Microsatellite instability and loss of heterozygosity in melanoma. J Invest Dermatol 105, 625-628 (1995)

96. A. LatiL, O. Cussenot, G. Fournier & R. Lidereau: The BRCA2 gene is not relevant to sporadic prostate tumours. Int J Cancer 66, 282-3 (1996)

97. A.K. El-Naggar, K. Hurr, J. Kagan, A. Gillenwater, D. Callender, M.A. Luna & H.J.G. Batsakis: Genotypic alterations in benign and malignant salivary gland tumors: histogenetic and clinical implications. Am J Surgical Pathol 21, 691-7 (1997)

98. M. Li, Z.F. Zhang, V.E. Reuter, C. Cordon-Cardo: Chromosome 3 allelic losses and microsatellite alterations in transitional cell carcinoma of the urinary bladder. Am J Pathol 149, 229-35 (1996)

99. G. Togo, N. Toda, F. Kanai, N. Kato, Y. Shiratori, K. Kishi, F. Imazeki, M. Makuuchi & M. Omata: A transforming growth factor beta type II receptor gene mutation common in sporadic cecum cancer with microsatellite instability. Cancer Res 56, 5620-23 (1996)

100. P.E. Berg, J. Liu, J. YinJ, M.G. Rhyu, C.N. Frantz & S.J. Meltzer: Microsatellite instability is infrequent in neuroblastoma. Cancer Epidemiol Biomark Prevent 4, 907-9 (1995)

101. E. Dams, E.J. van de Kelft, J.J. Martin, J. Verlooy & P.J. Willems: Instability of microsatellites in human gliomas. Cancer Res 55, 1547-9 (1995)

102. A.A. Larson, S. Kern, R.L. Sommers, J. Yokota, W.K. Cavenee & G.M. Hampton: Analysis of replication error (RER+) phenotypes in cervical carcinoma. Cancer Res 56, 1426-31 (1996)

103. A. Berchuck: Biomarkers in the endometrium. J Cell Biochem-Suppl 23, 174-8 (1995)

104. R. Wooster, A.M. Cleton-Jansen, N. Collins, J. Mangion, R.S. Cornelis, C.S. Cooper, B.A. Gusterson, B.A.J. Ponder, A. von Deimling, O.D. Wiestler, C.J. Cornelisse, P. Devilee & M.R. Straton: Instability of short tandem repeats (microsatellites) in human cancers. Nature Genet 6, 152-6 (1994)

105. C.J. Yee, N. Roodi, C.S. Verrier & F.F. Parl: Microsatellite instability and loss of heterozygosity in breast cancer. Cancer Res 54, 1641-4 (1994)

106. J.A. Shaw, T. Walsh, S.A. Chappell, N. Carey, K. Johnson & R.A. Walker: Microsatellite instability in early sporadic breast cancer. Br J Cancer 73, 1393-7 (1996)

107. T. Toyama, H. Iwase, H. Yamashita, H. Iwata, T. Yamashita, K. Ito, Y. Hara, M. Suchi, T. Kato, T. Nakamura & S. Kobayashi: Microsatellite instability in sporadic human breast cancers. Int J Cancer 68, 447-51 (1996)

108. T. Toyama, H. Iwase, H. Iwata, Y. Hara, Y. Omoto, M. Suchi, T. Kato, T. Nakamura & S. Kobayashi: Microsatellite instability in in situ and invasive sporadic breast cancers of Japanese women. Cancer Lett 108, 205-9 (1996)

109. J. I. Risinger, J.C. Barrett, P. Watson, H.T. Lynch & J. Boyd: Molecular genetic evidence of the occurrence of breast cancer as an integral tumor in patients with the hereditary nonpolyposis colorectal carcinoma syndrome. Cancer 7, 1836-43 (1996)

110. R.A. Walker, J.L. Jones, S. Chappell, T. Walsh & J.A. Shaw: Molecular pathology of breast cancer and its application to clinical management. Cancer Metastasis Rev 16, 5-27 (1997)

111. E.B. Rush, J.E. Calvano, K.J. van Zee, A.D. Zelenetz & P.I. Borgen: Microsatellite instability in breast cancer. Ann Surg Oncol 4, 310-5 (1997)

112. G. Souvinos, H. Kiaris, A. Tsikkinis, S. Vassilaros & D.A. Spandidos: Microsatellite instability and loss of heterozygosity in primary breast tumours. Tumor Biol 18, 157-66 (1997)

113. P. Karnik, S. Plummer,G. Casey, J. Myles, R. Tubbs, J. Crowe & B.R.G. Williams: Microsatellite instability at a single locus (D11S988) on chromosome 11p15.5 as a late event in mammary tumorigenesis. Hum Mol Genet 4, 1889-94 (1995)

114. C.M. Aldaz, T. Chen, A. Sahin, J. Cunningham & M. Bondy: Comparative allelotype of in situ and invasive human breast cancer: high frequency of microsatellite instability in lobular breast carcinomas. Cancer Res 55, 3976-81 (1995)

115. E.K. Dillon, W.B. de Boer,J.M. Papadimitriou & G.R. Turbett: Microsatellite instability and loss of heterozygosity in mammary carcinoma and its probable precursors. Br J Caner 76, 156-162(1997)

116. M. Kasami, C.L. Vnencak-Jones, S. Manning, W.D. Dupont & D.L. Page: Loss of heterozygosity and microsatellite instability in breast hyperplasia. Am J Pathol 150, 1925-32 (1997)

117. J.R. Eshleman & S.D. Markowitz: Mismatch repair defects in human carcinogenesis. Hum Mol Genet 5, 1489-94 (1996)

118. C.R. Boland: Roles of the DNA mismatch repair genes in colorectal tumorigenesis. Int J Cancer 69, 47-9 (1996)

1 19. Y. Huang, B. Bove, Y. Wu, I.H. Russo, Q. Tahin, X. Yang, A. Zekri & J. Russo: Carcinogenesis (submitted)

120. J.C. Murray, K.H. Buetow, J.L. Waber, S. Ludwigsen, T. Scherpbier-Heddema, F. Manion, J. Quillen, V.C. Sheffield, S. Sunden, G.M. Duyk, J. Weissenbach, G. Gyapay, C. Dib, J. Morrissette, G.M. Lathrop, A. Vignal, R. White, N. Matsunami, S. Gerken, R. Melis, H. Albertsen, R. Plaetke, S. Odelberg, D. Ward, J. Dausset, D. Cohen& H.Cann: A comprehensive human linkage map with centimorgan density. Science 265, 2049-2054 (1994)

121. Y. Wu, N. Barnabas, I.H. Russo, X. Yang & J. Russo: Microsatellite instability and loss of heterozygosity in chromosomes 9 and 16 in human breast epithelial cells transformed by chemical carcinogens. Carcinogenesis 18, 1069-74 (1997)

122. L.A. Loeb: Mutator phenotype may be required for multistage carcinogenesis. Cancer Res 51, 3075-9 (1991)

123. L.A. Loeb: Cancer cells exhibit a mutator phenotype. In: Advances in Cancer Research. Academic Press (1998) pp25-56.

124. E. Eisenstadt, A.J. Warren, J. Porter, D. Atkins & J.H. Miller: Carcinogenic epoxides of benzo[a]pyrene and cyclopenta[cd]pyrene induce base substitutions via specific transversions. Proc Natl Acad Sci USA 79,1945-9 (1982)

125. M.S. Greenblatt, W.P. Bennett, M. Hollstein & C.C. Harris: Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54:4855-78 (1994)

126. A. Prisieux, S. Lim, J. Groopman & M. Ozturk: Selective targeting of p53 gene mutation hotspots in human cells by etiologically defined carcinogens. Cancer Res 51, 6185-9 (1991)

127. D. Malkin: p53 and the Li-Fraumeni syndrome. Biochim Biophy Acta 1198, 197-213 (1994)

128. I. Mellon, D.K. Rajpal, M. Koi,C.R. Boland & G.N. Champe: Transcription-coupled repair deficiency and mutations in human mismatch repair genes. Science 272, 557-60 (1996)

129. E.A. Sia, S. Jinks-Robertson, T.D. Petes: Genetic control of microsatellite stability. Mutat Res 383, 61-70 (1997)

130. R.E.K. Fournier & F.H. Ruddle: Microcell-mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells. Proc Natl Acad Sci USA 74, 319-23 (1977)

131. E.J. Stanbridge: Genetic analysis of human malignancy using somatic cell hybrids and manochromosome transfer. Cancer Surv 7, 317-24 (1988)

132. M. Noda: Mechanisms of reversion. FASEB J 7, 834-846 (1993)

133. M. Oshimura: Lessons learned from studies on tumor suppression by microcell-mediated chromosome transfer. Environ Health Persp 93, 57-58 (1991)

134. M.J. Anderson & E.J. Stanbridge: Tumor suppressor genes studied by cell hybridization and chromosome transfer. FASEB J 7, 826-33 (1993)

135. J.D. Hunt: Evaluation of phenotypic alteration by microcell-mediated chromosome transfer. Analytical Biochem 238, 107-16 (1996)

136. B.E. Weissman, P.J. Saxon, S.R. Pasquale, G.R. Jones, A.G. Geiser & E.J. Stanbridge: Introduction of a normal human chromosome 11 into a Wilm’s tumor cell line controls its tumorigenic expression. Science 236, 175-80 (1987)

137. M. Oshimura, H. Kugoh, M. Koi, M. Shimuzu, H. Yamada, H. Satoh & J.C. Barrett: Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines. J Cell Biochem 42, 135-42 (1990)

138. H. Yamada, N. Wake, S. Fujimoto, J.C. Barrett & M. Oshimura: Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-medicated chromosome transfer. Oncogene 5, 1141-7 (1990)

139. K. Tanaka, M. Oshimura, R. Kikuchi, M. Seki, T. Hayashi & M. Miyaki: Suppression of tumorigenicity in human colon carcinoma cells by introduction of normal chromosome 5 or 18. Nature 349, 340-2 (1991)

140. X.W. Wang, X. Lin, C.B. Klein, R.K. Bhamra, Y.W. Lee & M. Costa: A conserved region in human and Chinese hamster X chromosome can induce cellular senescence of nickel-transformed Chinese hamster cell line. Carcinogenesis 13, 555-61 (1992)

141. T. Ichikawa, Y. Ichikawa, J. Dong, A.L. Howkins, C.A. Griffin, W.B. Isaacs, M. Oshimura, J.C. Barrett & J.T. Isaacs: Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome 11. Cancer Res 52, 3486-90 (1992)

142. M. Goyette, K. Cho, C.L. Fasching, D.B. Levy, K.W. Kinzler, C. Paraskeva, B. Vogelstein & E.J. Stanbridge: Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol Cell Bio 12, 1387-95 (1992)

143. G. Casey, S. Plummer, G. Hoeltge, D. Scanlon, C. Fasching, E.J. Stanbridge: Functional evidence for a breast cancer growth suppressor gene on chromosome 17. Hum Mol Genet 2, 1921-7 (1993)

144. H. Satoh, P.W. Lamb, J.T. Dong, H. Everitt, C. Boreiko, M. Oshimura &J.C. Barrett: Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosomes 3 and 11 introduced via microcell-mediated chromosome transfer. Mol Carcinog 7, 157-64 (1993)

145. D.R. Welsh, P. Chen, M.E. Miele, C.T. McGary, J.M. Bower, E.J. Stanbridge & B.E. Weissman: Microcell-mediated transfer of chromosome 6 into metastatic human C8161 melanoma cells suppresses metastasis but does not inhibit tumorigenicity. Oncogene 9, 255-62 (1994)

146. M. Negrini, S. Sabbioni, S. Haldar, L. Possati, A. Castagnoli, A. Corallini, G. Barbanti-Brodano & C.M. Croce: Tumor and growth suppression of breast cancer cells by chromosome 17-associated functions. Cancer Res 54, 1818-24 (1994)

147. M. Negrini, S. Sabbioni, L. Possati, S. Rattan, A. Corallini, G. Barbanti-Brodano & C.M. Croce: Suppression of tumorigenicity of breast cancer cells by microcell-mediated chromosome transfer: studies on chromosomes 6 and 11. Cancer Res 54: 1331-6 (1994)

148. M.A. Yoshida, M. Shimizu, T. Ikeuchi, A. Tonomura, J. Yokota & M. Oshimura: In vitro growth suppression and morphological change in a human renal cell carcinoma cell line by the introduction of normal chromosome 3 via microcell fusion. Mol Carcinog 9, 114-21 (1994).

149. A. Yoshida, T. Asaga, C. Masuzawa & S. Kawahara: Alteration of tumorigenicity in undifferentiated thyroid carcinoma cells by introduction of normal chromosome 11. J Surg Oncol 55, 170-4 (1994)

150. M.J. Anderson, G. Casey, C.L. Gasching & E.J. Stanbridge: Evidence that wild-type TP53, and not genes on either chromosome 1 or 11, controls the tumorigenic phenotype of the human fibrosarcoma HT1080. Gene Chrom Cancer 9, 266-81 (1994)

151. Y.S. Murakami, A.R. Brothman, R.J. Leach & R.L. White: Suppression of malignant phenotype in a human prostate cancer cell line by fragments of normal chromosomal region 17q. Cancer Res 55, 3389-94 (1995)

152. M. Theile, Z. Seitz, W. Arnold, B. Jandrig, R. Frege, P.M. Schlag, W. Haensch, H. Guski, K.J. Winzer, J.C. Barrett & S. Scherneck: A defined chromosome 6q fragment (at D6S310) harbors a putative tumor suppressor gene for breast cancer. Oncogene 13, 677-85 (1996)

153. N. Uzawa, M.A. Yoshida, M. Oshimura & T. Ikeuchi: Suppression of tumorigenicity in three different cell lines of human oral squamous cell carcinoma by introduction of chromosome 3p via microcell-mediated chromosome transfer. Oncogene 11, 1997-2004 (1995)

154. S.J. Plummer, L. Adams, J.A. Simmons & G. Casey: Localization of a growth suppressor activity in MCF7 breast cancer cells to chromosome 17q24-q25. Oncogene 14, 2339-45 (1997)

155. M. Oshimura, M. Shimizu & H. Kugoh: Genetic regulation of telomerase in a multiple pathways model to cellulose senescence. Hum Cell 9, 301-8 (1996)

156. K.G. Klein & N.P. Bouck: The distal region of the long arm of human chromosome 1 carries tumor suppressor activity for a human fibrosarcoma line. Cancer Genet Cytogenet 73, 109-21 (1994)

157. I. Horikawa, H. Yamada, H. Kugoh, Y. Yuasa, M. Suzuki & M. Oshimura: Subchromosomal mapping of a putative transformation suppressor gene on human chromosome 1. Jpn J Cancer Res 86, 444-50 (1995)

158. M. Shimizu, J. Yokota, N. Mori, T. Shuin, M. Shinoda, T. Masaaki & M. Oshimura: Introduction of normal chromosome 3p modulates the tumorigenicity of a human renal cell carcinoma cell line YCR. Oncogene 5, 185-94 (1990)

159. P. Remessi, F. Gualandi, C. Morlli, C. Trabanelli, Q. Wu, L. Possati, M. Montesi, J.C. Barrett & G. Barbanti-Brodano: Transfer of human chromosome 3 to an ovarian carcinoma cell line identifies three regions on 3p involved in ovarian cancer. Oncogene 9, 3467-74 (1994)

160. M. Koi, H. Morita, H. Yamada, H. Satoh, J.C. Barrett & M. Oshimura: Normal human chromosome 11 suppressed tumorigenicity human cervical tumor cell line SiHa. Mol Carcinog 2, 12-21 (1989)

161. G. Robertson, A. Coleman & T.G. Lugo: A malignant melanoma tumor suppressor on human chromosome 11. Cancer Res 56, 4487-92 (1996)

162. A.L. Menke, R.C. van Ham, E. Sonneveld, A. Shvarts, E.J. Stanbridge, K. Miyagawa, A.J. van der EB & A.G. Jochemsen: Human chromosome 11 suppresses the tumorigenicity of adenovirus transformed baby rat kidney cells: involvement of the Wilm’s tumor 1 gene. Int J Cancer 63, 76-85 (1995)

163. M. Theile, S. Hartmann, H. Naundorf, D.R. Birgitelbe, H. Krause, W. Deppert, J.C. Barrett & S. Scherneck: Wild-type p53 is not involved in reversion of the tumorigenic phenotype of breast cancer cells after transfer of normal chromosome 17. Int J Oncol 4, 1067-75 (1994)

164. A. Umar, M. Koi, J.I. Risinger, W.E. Glaab, K.R. Tindal, R.D. Kolodner, C.R. Boland, J.C. Barrett & T.A. Kundel: Correction of hypermutability, N-methyl-N’-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6. Cancer Res 57, 3949-55 (1997)

165. D.J. Chen, B.L. Marrone, T. Nguyen, M. Stackhouse, Y. Zhao, M.J. Siciliano: Regional assignment of a human DNA repair gene (XRCC5) to 2q34 by X-ray hybrid mapping. Genomics 21, 423-7 (1992)

166. M. Koi, A. Umar, D.P. Chauhan, S.P. Cherian, J.M. Carethers, T.A. Kunkel & C.R. Boland: Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N’-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation. Cancer Res 54, 4308-12 (1994)

167. A. Kurimasa, Y. Nagata, M. Shimizu, E. Mitsuru, Y. Nakamura & M. Oshimura: A human gene that restores the DNA-repair defect in SCID mice is located on 8p11.1-q11.1. Hum Genet 93, 21-6 (1994)

168. S. Kodama, K. Komatsu, Y. Okumura & M. Oshimura: Suppression of X-ray-induced chromosome aberration in ataxia telangiectasia cells by introduction of a normal human chromosome 11. Mutation Res 293, 31-7 (1992)

169. Y. Ejima, M. Oshimura, M.S. Sassaki: Determination of the chromosomal site for the human radiosensitive ataxia telangiectasia gene by chromosome transfer. Mutation Res 250, 337-43 (1991)

170. T. Ichikawa, N. Nihei, H. Kuramochi, Y. Kawana, A.M. Killary, C.W. Rinker-Scharffer, J.C. Barrett, J.T. Isaacs, H. Kugoh, M. Oshimura & J. Shimazaki: Metastasis suppressor genes for prostate cancer. Prostate-Suppl 6, 31-5 (1996)

171. N. Nihei, T. Ichikawa, Y. Kawana, H. Kuramochi, H. Kugoh, M. Oshimura, I. HayataI, J. Shimazaki & H. Ito: Mapping of metastasis suppressor gene(s) for rat prostate cancer on the short arm of human chromosome 8 by irradiated microcell-mediated chromosome transfer. Gene Chrom Cancer 17, 260-8 (1995)

172. K.K. Phillips, D.R. Welsh, M.E. Miele, J.H. Lee & B.E. Weissman: Suppression of MDA-MB-435 breast carcinoma cell metastasis following the introduction of human chromosome 11. Cancer Res 56, 1222-7 (1996)

173. C.W. Rinker-Schaeffer, A.L. Hawkins, N. Ru, J. Dong, G. Stoica, C.A. Griffin, T. Ichikawa, J.C. Barrett & J.T. Isaacs: Differential suppression of mammary and prostate cancer metastasis by human chromosomes 17 and 11. Cancer Res 54, 6249-56 (1994)

174. X. Yang, Y. Huang, I.H. Russo, J.C. Barrett and J. Russo: Chromosomes 11 and 17 revert transformed phenotypes induced by benzo(a)pyrene (BP) in human breast epithelial cells (HBEC). Proc Am. Assoc. Cancer Res 39: 185 (1998)

175. J. Taylor-Papadimitriou, F. Berdichevsky, B. D’Souza & J. Burchell: Human models of breast cancer. Cancer Surveys 16, 59-78 (1993)

176. T. Kuroki & N.-H. Huh: Why are human cells resistant to malignant cell transformation in vitro. Jpn J Cancer Res 84, 1091-100 (1993)