[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

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.