[Frontiers in Bioscience 2, a18-25, July 15, 1997]
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DISTINCT TUMOR SPECIFIC EXPRESSION OF TGFB4 (ebaf)*, A NOVEL HUMAN GENE OF THE TGF-b SUPERFAMILY

Siamak Tabibzadeh, Ravi Kothapalli , Ibrahim Buyuksal

Dept of Pathology, H. Lee Moffitt Cancer Center at the University of South Florida, College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612

Received 7/10/97 Accepted 7/15/97 *: Patent pending

4. RESULTS

We first examined the expression of the TGFB4 (ebaf) mRNA in normal tissues (Fig 1, Table 1). RNAs from several normal late secretory and menstrual endometrial tissues were used as control in these experiments (Fig 1-5). As reported previously (15), the prominent TGFB4 (ebaf) mRNA exclusively expressed in endometrium during the late secretory and menstrual phases was 2.5 kb (Fig 1). However, additional, smaller in size mRNAs were also noted in the menstrual endometria (Fig 3, Fig 5). These mRNAs were 2.1 and 1.5 kb in size (Fig 3, Fig 5). From a large number of tissues tested, the TGFB4 (ebaf) mRNA was expressed only in the ovary, rectum, testis and pancreas (Fig 1, Table 1). Both 2.5 and 2.1 kb TGFB4 (ebaf) mRNAs were expressed in the pancreas, whereas the TGFB4 (ebaf) mRNA, weakly expressed, in the rectum, in an ovary and testis, was 2.1 kb (Fig 1, Table 1). TGFB4 (ebaf) mRNA was not expressed in the breast, stomach, small bowel, colon, kidney, lung, fallopian tube, spleen and lymph node (Fig 1, Table 1).

Fig 1. Northern blot analysis of TGFB4 (ebaf) mRNA in normal tissues. 20 mg total RNA from each tissue (lane 1: normal menstrual endometrium serving as the positive control) and other normal tissues (lane 2: spleen, lane 3: lymph node, lanes 4 and 5: stomach, lane 6: lung, lane 7: breast, lane 8: liver, lane 9 and 10: ovary, lane 11: rectum, lane 12: testis, lane 13: pancreas) was subjected to the Northern blot analysis using the entire placental-derived TGFB4 (ebaf) cDNA as the probe (upper panel). The integrity of RNA and equal loading was verified by staining the 18S and 28S ribosomal RNAs (not shown) and hybridization of the blots with a cDNA probe to GAPDH (lower panel). As shown, bands of TGFB4 (ebaf) mRNA in the size of 2.1 and 2.5 kilobase (kb) are detected in the endometrium. A weak 2.1 kb TGFB4 (ebaf) mRNA is detected in the ovary, rectum, and testis. In the pancreas, both the 2.1 and 2.5 kb TGFB4 (ebaf) mRNA are detected.

Table 1. Expression of TGFB4 (ebaf) mRNA in normal human tissues.

TISSUE

NUMBER OF TISSUES

NORTHERN BLOT FINDING

Breast

5

-

Stomach

3

-

Small Bowel

1

-

Colon

11

-

Rectum

2

2.1 kb

Liver

6

-

Pancreas

2

2.1 and 2.5 kb

Kidney

1

-

Lung

1

-

Fallopian Tube

1

-

Ovary

7

-

Ovary

1

2.1 kb

Testis

2

2.1 kb

Spleen

1

-

Lymph node

1

-

We then examined the TGFB4 (ebaf) mRNA expression in the cancers derived from cells of different lineages. In eleven adenocarcinomas of colon, adjacent normal colonic tissues, non-involved by the tumor were available for the study. The RNAs from the neoplastic and surrounding normal tissues were both subjected to the Northern blot analysis for the detection of the TGFB4 (ebaf) mRNA (Fig 2, Table 2).

Figure 2. Northern blot analysis of TGFB4 (ebaf) mRNA in colonic adenocarcinomas. 20 mg total RNAs from a normal late secretory endometrium which served as the positive control (lane 1) as well as mucinous adenocarcinomas of colon (lanes 2, 4, 6), non-mucinous adenocarcinomas of colon (lanes 8, 10, and 12) and adjacent normal colon (lanes 3, 5, 7, 9, 11 and 13) were subjected to the Northern blot analysis using the entire placental-derived TGFB4 (ebaf) cDNA as the probe (upper panel). A 2.1 kb TGFB4 (ebaf) mRNA is detected in the colonic adenocarcinomas with mucinous differentiation and not those which did not exhibit a mucinous differentiation or the adjacent normal colonic tissues. The integrity of RNA and equal loading was verified by staining the 18S and 28S ribosomal RNAs (not shown) and hybridization of the blots with a cDNA probe to GAPDH (lower panel).

Table 2. Expression of TGFB4 (ebaf) mRNA in human tumors

TUMOR TYPE

NUMBER OF TUMORS

NORTHERN BLOT FINDING

Mucinous adenocarcinoma of colon*

7

2.1 kb

Mucinous adenocarcinoma of colon*

4

-

Mucinous adenocarcinoma of the duodenum

1

2.1 kb

Mucinous adenocarcinoma of the ovary

3

2.1 kb

Serous cystadenocarcinoma of ovary

2

2.1 kb

Serous cystadenocarcinoma of ovary

2

2.5 kb

Serous cystadenocarcinoma of ovary

5

-

Non-mucinous colonic adenocarcinoma metastatic to ovary

1

-

Non-mucinous adenocarcinoma of colon

7

-

Non-mucinous adenocarcinoma of uterine cervix

1

-

Non-mucinous adeoncarcinoma of the stomach

3

-

Endometrioid adenocarcinoma of ovary

1

-

Hepatocelluar carcinoma

3

-

Renal Cell Carcinoma

3

-

Liver metastasis; consistent with colonic primary*

6

-

Adenocarcinoma of lung

7

-

Adenocarcinoma of breast*

5

-

Adenocarcinoma of endometrium

3

-

SCC of the Larynx

1

-

SCC of the Lung

4

-

SCC of the Uterine cervix

1

-

Teratoma-embryonal cell carcinoma

1

2.5 kb

Germ cell tumor-embryonal cell carcinoma

1

2.5 kb

Seminoma

2

2.1 kb

Seminoma

1

-

Leiomyosarcoma, gastric

1

-

Leiomyosarcoma, colon

1

-

Leiomyosarcoma, pelvic

1

-

Chondrosarcoma, thoracic wall

1

-

Osteosarcoma, metastatic to the lung

3

-

Liposarcoma, retroperitoneum

1

-

Synovial sarcoma, metastatic to the chest wall

1

-

Synovial sarcoma, parotid

1

-

Synovial sarcoma, leg

1

-

Angiosarcoma, mediastinal

1

-

Lymphoma

1

-

Lymphoma, B cell type

1

-

Lymphoma, B cell, spleen

1

-

Lymphoma, T cell, groin

1

-

Lymphoma, T cell, angiocentric, hip

1

-

Hodgkinís disease, mixed cell type, lymph node

1

-

Melanoma

5

-

*: Normal tissues around the tumors were available for the Northern blot analysis and did not exhibit TGFB4 (ebaf) mRNA

Whereas the TGFB4 (ebaf) mRNA was not detected in the normal colon, the expression of the 2.1 kb TGFB4 (ebaf) mRNA was detected in seven of the eleven cases of adenocarcinomas of colon (Fig 2, Table 2). The histologic evaluation of the positive cases revealed these cases to have a mucinous differentiation. Similarly, an adenocarcinoma of the duodenum and three cases of mucinous adenocarcinomas of the ovary that also exhibited mucinous differentiation expressed the 2.1 kb TGFB4 (ebaf) mRNA (Fig 3, Table 2).

Figure 3. Northern blot analysis of TGFB4 (ebaf) mRNA in ovarian adenocarcinomas. 20 mg total RNA from a normal menstrual endometrium which served as the positive control (lane 1) as well as other tumors (lanes 2-4, serous cystadenocarcinomas; lane 5: endometrioid adenocarcinoma; lane 6, mucinous cystadenocarcinoma) was subjected to the Northern blot analysis using the entire placental-derived TGFB4 (ebaf) cDNA as the probe (upper panel). There is relatively more TGFB4 (ebaf) mRNAs in the menstrual endometrium than the tumor tissues leading to overexposure of the band of TGFB4 (ebaf) mRNAs in endometrium. The 2.5 kb TGFB4 (ebaf) mRNA is detected in serous (lanes 3 and 4) and mucinous cystadenocarcinomas (lane 6) of the ovary. The 2.1 kb mRNA is detected in serous cystadenocarcinoma (lane 2). The endometrioid adenocarcinoma of the ovary does not exhibit TGFB4 (ebaf) mRNA. The integrity of RNA and equal loading was verified by staining the 18S and 28S ribosomal RNAs (not shown) and hybridization of the blot with a cDNA probe to GAPDH (lower panel).

The non-mucinous adenocarcinomas, whether primary in the colon, metastatic to the liver, or the ovary, did not express TGFB4 (ebaf) mRNA (Table 2). In addition, in colonic adenocarcinomas metastatic to the liver, the adjacent liver, non-involved by the tumor, also did not show any evidence of TGFB4 (ebaf) mRNA expression (Table 1). In the ovary, besides the mucinous adenocarcinomas some serous adenocarcinomas (n=4/9) also expressed the TGFB4 (ebaf) mRNA (Table 2). In two cases, the TGFB4 (ebaf) mRNA was 2.1 and in two cases 2.5 kb (Fig 3, Table 2). On the other hand, other adenocarcinomas of ovary including those exhibiting endometrioid differentiation failed to express TGFB4 (ebaf) mRNA (Fig 3). To localize the cells that express the TGFB4 (ebaf) mRNA in the adenocarcinomas of colon and ovary, in situ hybridization was carried out on the positive cases. As a positive control, tissue sections from a late secretory endometrium, was used. As reported previously (15), the hybridization signal was noted primarily in the endometrial stroma and endometrial glands or the endothelium did not show presence of signal (Fig 4A). Only rarely, few glands close to the surface epithelium exhibited a positive hybridization signal (data not shown). Hybridization with the sense RNA did not produce any signal in the normal endometrium (Fig 4B). In tumor tissue sections hybridized with the anti-sense TGFB4 (ebaf) RNA, there was some minimal expression of TGFB4 (ebaf) mRNA in the tumor stroma. However, by far the most prominent expression of TGFB4 (ebaf) mRNA was noted in the neoplastic epithelial cells both in the colon (Fig 4C) and in the ovary (Fig 4E). The sense probe of the TGFB4 (ebaf) failed to show a hybridization signal in the same tissues (Fig 4D, 5F). In the adenocarinomas of other organs such as breast, lung, pancreas, cervix, stomach, liver, kidney and endometrium, TGFB4 (ebaf) mRNA was not detectable by the Northern blot analysis (Table 2).

Fig 4. In situ hybridization of TGFB4 (ebaf) mRNA in adenocarcinomas of colon and ovary. Digoxigenin-labeled anti-sense (A, C, E) and sense RNAs (B, D, F) of TGFB4 (ebaf) were synthesized by in vitro transcription of the full length TGFB4 (ebaf) cDNA cloned into pBluescriptR SK(+/-). Sections of a late secretory endometrium were used as the positive control (A). Hybridization signal is seen in the endometrial stroma (A) and in the epithelial cells of a colonic adenocarcinoma with mucinous differentiation (C) and ovarian adenocarcinoma (E). The sense probe did not reveal any hybridization signal in the sections of the endometrium (B) or the same tumors (D, F).

Five cases of testicular cancer were examined for TGFB4 (ebaf) mRNA expression by the Northern blot analysis. Two cases that showed an embryonal carcinoma component exhibited the 2.5 kb TGFB4 (ebaf) mRNA. On the other hand, two out of three cases of seminomas expressed the 2.1 kb TGFB4 (ebaf) mRNA (Fig 5).

Fig 5. Northern blot analysis of TGFB4 (ebaf) mRNA in testicular cancers. 20 mg total RNA from a normal menstrual endometrium which served as the positive control (lane 1) and each tumor tissue (lane 2: teratoma-embryonal cell carcinoma, lane 3: mixed germ cell tumor containing embryonal carcinoma, lanes 4-6; seminoma) was subjected to the Northern blot analysis using the entire placental-derived TGFB4 (ebaf) cDNA as the probe (upper panel). ). The blot was exposed for long duration to detect TGFB4 (ebaf) mRNA in the neoplastic tissues. This resulted in the overexposure of the TGFB4 (ebaf) mRNAs detected in the endometrium. The 2.5 kb TGFB4 (ebaf) mRNA is detected in the tumors containing embryonal carcinoma. The 2.1 kb mRNA is detected in two of three cases of seminoma. The integrity of RNA and equal loading was verified by staining the 18S and 28S ribosomal RNAs (not shown) and hybridization of the blots with a cDNA probe to GAPDH (lower panel).

The Northern blot analysis of squamous cell carcinomas derived from larynx, lung and uterine cervix did not show any evidence of the TGFB4 (ebaf) mRNA expression (Table 2). Furthermore, the TGFB4 (ebaf) mRNA was not detectable in the non-epithelial tumors including sarcomas such as leiomyosarcoma, chondrosarcoma, osteosarcoma, liposarcoma, synovial sarcoma, as well as Hodgkin's and non-Hodgkin's lymphomas, or melanomas (Table 2).