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Allelic losses and oncogene amplification in human ovarian carcinomas of different histopathologic grades
184
SOCIETY OF GYNECOLOGISTS
and are maintained in monolayer culture. By Northern analysis of total RNA isolated from these cells, we found that the level of expression of CNX43 mRNA increased with the degree of confluence and with time in culture. Using serum as a mitogen, i.e., stimulus for these cells, we found that there was an inverse relationship between the level of CNX43 mRNA expression and DNA synthesis (i.e., [‘Hlthymidine incorporation into trichloracetic acid-precipitable material). Interleukin1 (IL-l), a cytokine that we have shown previously to act on endometrial stromal cells to stimulate synthesis of IL-l, IL-6, and prostaglandins, acts on these cells to inhibit synthesis of CNX43 mRNA. The inhibition occurs in a time- and concentration-dependent manner. IL-l, however, does not stimulate [“Hlthymidine incorporation into these cells. Thus, endometrial stromal cells respond to selected stimuli of mitogenesis by a decrease in CNX43 mRNA expression. Based on these data, however, we suggest that IL-1 may serve a role in regulation of GJ synthesis independent of mitogenesis. Connexin43 expression and the regulation of intercellular communication through GJs may represent biochemical mechanisms involved in the replication of neoplastic endometrial cells. 68. Allelic Losses and Oncogene Amplification in Human Ovarian Carcinomas of Different Histopathologic Grades. W. ROBINSON, AND L.
DUBEAU,University of Southern California, Los Angeles, California 90033. Nonrandom losses of genetic material from specific chromosomal segments have been documented in a variety of malignant tumors. Such losses, which may result in the inactivation of genetic suppressors of the malignant phenotype have recently been shown on chromosomal segments 3p, 6q, lip, and 17p in human ovarian carcinomas. In the present study we used recombinant DNA technologies to examine the frequencies of losses of heterozygosity involving loci from the above chromosomes as well as from chromosomal segments 5p, 10, 13q, and 21q in 29 ovarian carcinomas. Cloned DNA fragments complementary to sequences showing restriction fragment length polymorphisms were used to identify losses in tumor DNA after determining which alleles were present in DNA extracted from normal leukocytes of the same subjects. Losses of heterozygosity were found on chromosomal segments 3p, 6q, llp, and 17p in 30, 43, 33, and 22% of patients, respectively. Loci from the remaining chromosomes showed losses in less than 15% of cases and 2 of them, 5p and 13q, showed no losses in the tumors examined. This indicates that the above findings on choromosomes 3, 6, 11, and 17 were nonrandom and that loci from these chromosomes are probably important in ovarian carcinogenesis. No genetic amplification of the protooncogenes H-ras, K-ras, or c-myc could be detected in any of the tumors examined. The tumors were graded histopathologically according to FIG0 guidelines and grouped according to molecular findings. Grade III (poorly differentiated) tumors were found to have greater numbers of allelic losses than tumors of grades I or II. 69. Ovarian Cancer Gene Expression: Differential Expression of Genes in Cancer and Normal Ovarian Surface Epithelium. Y. J. LIWR,
M-F. PFENNINGER, AND E. BRAND, University of Colorado Health Sciences Center, Denver, Colorado 80262. It is unclear whether new proteins are expressed during the malignant transformation of ovarian epithelium. Previous studies have not demonstrated genes or gene products unique to ovarian cancer. Based on preliminary work in which, by two-dimensional gel electrophoresis, we identified unique proteins in epithelial ovarian cancer (OVCA) that were not found in normal surface epithelium (OSE) cell lines, the present study attempts to find the complimentary DNA (cDNA) encoding for these proteins. The mRNAs of five normal OSE cell lines and five established OVCA were isolated. These mRNAs were pooled and used to synthesize “normal OSE” and “malignant OVCA” cDNA libraries, using pcDNA I phagemid as the recombinant cloning vector and Escherichia coli MC1061/P3 as the host. Differential screening of the bacterial colonies in the OSE and OVCA cDNA libraries has dem-
ONCOLOGISTS-ABSTRACTS onstrated several recombinant phagemids carrying cDNA clones unique to OVCA. The resulting clones made up a subtraction cDNA library highly enriched with ovarian cancer distinctive genes (OCDG). The nucleic acid sequences of the OCDG will be subsequently analyzed to determine whether they encode for the ovarian cancer unique proteins identified. Further studies should shed light on the role played by OCDG in the malginant transformation of the normal surface epithelium. 70. Identification of HPV 31 B in Human Cervical Cells Grown on Raised Collagen Gel Rafts. G. WILBANKS,* M. TURYK,* AND M. HOSKEN,*
M. BEDELL,? J. HUDSON,? T. GOLuB,t AND L. LAIMINS,t Presbyterian-St. Luke’s Medical Center; and tuniversity cago, Chicago, Illinois 60637.
*Rushof Chi-
Cells derived from a biopsy of early cervical intraepithelial neoplasia have been cultured and passaged in tissue culture. These cells exhibited an extended lifespan as compared to normal human ectocervical keratinocytes and have been in culture now for 16 months. When transferred to collagen gel rafts raised to the air/liquid interface, the CIN612 cells exihibited an expanded basal layer as well as some perinuclear clearing and abnormal nuclei (koilocytosis) in the upper layers of the epithelium similar in histological appearance to the biopsy from which the proliferation cells were derived. Episomal HPV 31 DNA was detected at approximately 50 copies per cell in CIN-612 cells. The HPV 31 DNA was shown to contain several unique restriction endonuclease sites and was, therefore, designated HPV 31b. In situ hybridization of CIN-612 collagen gel rafts with a HPV 31 type-specific DNA probe demonstrated the amplification of HPV-31 DNA in the upper layers of the epithelium. 71. ‘H-Labeled Subgenomic mRNA Probes Can Demonstrate Subtle Differences in Human Papilloma Virus Expression in Preinvasive and Invasive Vulvar Cancers. J. S. RADER, J. S. PARK, T. C. WV, K. V.
R. J. KURMAN, L. A. LAIMINS, AND J. L. CURRIE, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205.
SHAH,
Demonstration of HPV DNA segments in cervical and vulvar neoplasias has become commonplace and led to widespread belief that HPV may be etiologically responsible for these malignancies. Recent work from this institution demonstrated clear differences in HPV 16 expression in vulvar cancers, showing heterogeneity in different age groups and histologic variants of patients with vulvar cancer. We have employed in situ hybridization for detection of subgenomic transcripts and immunostaining for Ll capsid protein to elucidate the role of HPV type 16 in vulvar cancer. These analyses revealed that the transcripts of E6E7 region were more abundant than those in Ll region in vulvar neoplastic tissues. The transcripts from early and late regions of HPV-16 continued to increase with the differentiation of the epithelial cells in both the warty and the basaloid types of vulvar precancerous lesions. In the current study, ‘H-labeled subgenomic probes were used to explore differences in viral mRNA expression. Formalin-fixed, paraffin-embedded tissue blocks of HPV-16-positive vulvar specimens were screened for subgenomic HPV-16 transcripts and capsid antigen. In situ hybridization was done with ‘H-labeled single-stranded RNA probes of the E6-E7, and Ll open reading frames of HPV-16. To verify specificity, both antisense and sense probes were used. In addition, epithelial signal intensity was always compared to the background stromal signal. Ll capsid protein was detected by immunostaining. The early and late RNA transcripts were analyzed in relation to morphological characteristics, cellular differentiation, and capsid protein production. The transcripts from the E6-E7 region were more abundant than those of Ll in vulvar neoplastic lesions. All subgenomic transcripts were most prominent in the terminally differentiated epithelium of both the warty and basaloid type intraepithelial lesions. This pattern of high-intensity signal in the most superficial epithelium was also seen in warty cancer. In contrast to cervical lesions, transcripts from the Ll region and viral capsid proteins were detected in a small subset of invasive vulvar cancer cells.
SOCIETY OF GYNECOLOGISTS
and are maintained in monolayer culture. By Northern analysis of total RNA isolated from these cells, we found that the level of expression of CNX43 mRNA increased with the degree of confluence and with time in culture. Using serum as a mitogen, i.e., stimulus for these cells, we found that there was an inverse relationship between the level of CNX43 mRNA expression and DNA synthesis (i.e., [‘Hlthymidine incorporation into trichloracetic acid-precipitable material). Interleukin1 (IL-l), a cytokine that we have shown previously to act on endometrial stromal cells to stimulate synthesis of IL-l, IL-6, and prostaglandins, acts on these cells to inhibit synthesis of CNX43 mRNA. The inhibition occurs in a time- and concentration-dependent manner. IL-l, however, does not stimulate [“Hlthymidine incorporation into these cells. Thus, endometrial stromal cells respond to selected stimuli of mitogenesis by a decrease in CNX43 mRNA expression. Based on these data, however, we suggest that IL-1 may serve a role in regulation of GJ synthesis independent of mitogenesis. Connexin43 expression and the regulation of intercellular communication through GJs may represent biochemical mechanisms involved in the replication of neoplastic endometrial cells. 68. Allelic Losses and Oncogene Amplification in Human Ovarian Carcinomas of Different Histopathologic Grades. W. ROBINSON, AND L.
DUBEAU,University of Southern California, Los Angeles, California 90033. Nonrandom losses of genetic material from specific chromosomal segments have been documented in a variety of malignant tumors. Such losses, which may result in the inactivation of genetic suppressors of the malignant phenotype have recently been shown on chromosomal segments 3p, 6q, lip, and 17p in human ovarian carcinomas. In the present study we used recombinant DNA technologies to examine the frequencies of losses of heterozygosity involving loci from the above chromosomes as well as from chromosomal segments 5p, 10, 13q, and 21q in 29 ovarian carcinomas. Cloned DNA fragments complementary to sequences showing restriction fragment length polymorphisms were used to identify losses in tumor DNA after determining which alleles were present in DNA extracted from normal leukocytes of the same subjects. Losses of heterozygosity were found on chromosomal segments 3p, 6q, llp, and 17p in 30, 43, 33, and 22% of patients, respectively. Loci from the remaining chromosomes showed losses in less than 15% of cases and 2 of them, 5p and 13q, showed no losses in the tumors examined. This indicates that the above findings on choromosomes 3, 6, 11, and 17 were nonrandom and that loci from these chromosomes are probably important in ovarian carcinogenesis. No genetic amplification of the protooncogenes H-ras, K-ras, or c-myc could be detected in any of the tumors examined. The tumors were graded histopathologically according to FIG0 guidelines and grouped according to molecular findings. Grade III (poorly differentiated) tumors were found to have greater numbers of allelic losses than tumors of grades I or II. 69. Ovarian Cancer Gene Expression: Differential Expression of Genes in Cancer and Normal Ovarian Surface Epithelium. Y. J. LIWR,
M-F. PFENNINGER, AND E. BRAND, University of Colorado Health Sciences Center, Denver, Colorado 80262. It is unclear whether new proteins are expressed during the malignant transformation of ovarian epithelium. Previous studies have not demonstrated genes or gene products unique to ovarian cancer. Based on preliminary work in which, by two-dimensional gel electrophoresis, we identified unique proteins in epithelial ovarian cancer (OVCA) that were not found in normal surface epithelium (OSE) cell lines, the present study attempts to find the complimentary DNA (cDNA) encoding for these proteins. The mRNAs of five normal OSE cell lines and five established OVCA were isolated. These mRNAs were pooled and used to synthesize “normal OSE” and “malignant OVCA” cDNA libraries, using pcDNA I phagemid as the recombinant cloning vector and Escherichia coli MC1061/P3 as the host. Differential screening of the bacterial colonies in the OSE and OVCA cDNA libraries has dem-
ONCOLOGISTS-ABSTRACTS onstrated several recombinant phagemids carrying cDNA clones unique to OVCA. The resulting clones made up a subtraction cDNA library highly enriched with ovarian cancer distinctive genes (OCDG). The nucleic acid sequences of the OCDG will be subsequently analyzed to determine whether they encode for the ovarian cancer unique proteins identified. Further studies should shed light on the role played by OCDG in the malginant transformation of the normal surface epithelium. 70. Identification of HPV 31 B in Human Cervical Cells Grown on Raised Collagen Gel Rafts. G. WILBANKS,* M. TURYK,* AND M. HOSKEN,*
M. BEDELL,? J. HUDSON,? T. GOLuB,t AND L. LAIMINS,t Presbyterian-St. Luke’s Medical Center; and tuniversity cago, Chicago, Illinois 60637.
*Rushof Chi-
Cells derived from a biopsy of early cervical intraepithelial neoplasia have been cultured and passaged in tissue culture. These cells exhibited an extended lifespan as compared to normal human ectocervical keratinocytes and have been in culture now for 16 months. When transferred to collagen gel rafts raised to the air/liquid interface, the CIN612 cells exihibited an expanded basal layer as well as some perinuclear clearing and abnormal nuclei (koilocytosis) in the upper layers of the epithelium similar in histological appearance to the biopsy from which the proliferation cells were derived. Episomal HPV 31 DNA was detected at approximately 50 copies per cell in CIN-612 cells. The HPV 31 DNA was shown to contain several unique restriction endonuclease sites and was, therefore, designated HPV 31b. In situ hybridization of CIN-612 collagen gel rafts with a HPV 31 type-specific DNA probe demonstrated the amplification of HPV-31 DNA in the upper layers of the epithelium. 71. ‘H-Labeled Subgenomic mRNA Probes Can Demonstrate Subtle Differences in Human Papilloma Virus Expression in Preinvasive and Invasive Vulvar Cancers. J. S. RADER, J. S. PARK, T. C. WV, K. V.
R. J. KURMAN, L. A. LAIMINS, AND J. L. CURRIE, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205.
SHAH,
Demonstration of HPV DNA segments in cervical and vulvar neoplasias has become commonplace and led to widespread belief that HPV may be etiologically responsible for these malignancies. Recent work from this institution demonstrated clear differences in HPV 16 expression in vulvar cancers, showing heterogeneity in different age groups and histologic variants of patients with vulvar cancer. We have employed in situ hybridization for detection of subgenomic transcripts and immunostaining for Ll capsid protein to elucidate the role of HPV type 16 in vulvar cancer. These analyses revealed that the transcripts of E6E7 region were more abundant than those in Ll region in vulvar neoplastic tissues. The transcripts from early and late regions of HPV-16 continued to increase with the differentiation of the epithelial cells in both the warty and the basaloid types of vulvar precancerous lesions. In the current study, ‘H-labeled subgenomic probes were used to explore differences in viral mRNA expression. Formalin-fixed, paraffin-embedded tissue blocks of HPV-16-positive vulvar specimens were screened for subgenomic HPV-16 transcripts and capsid antigen. In situ hybridization was done with ‘H-labeled single-stranded RNA probes of the E6-E7, and Ll open reading frames of HPV-16. To verify specificity, both antisense and sense probes were used. In addition, epithelial signal intensity was always compared to the background stromal signal. Ll capsid protein was detected by immunostaining. The early and late RNA transcripts were analyzed in relation to morphological characteristics, cellular differentiation, and capsid protein production. The transcripts from the E6-E7 region were more abundant than those of Ll in vulvar neoplastic lesions. All subgenomic transcripts were most prominent in the terminally differentiated epithelium of both the warty and basaloid type intraepithelial lesions. This pattern of high-intensity signal in the most superficial epithelium was also seen in warty cancer. In contrast to cervical lesions, transcripts from the Ll region and viral capsid proteins were detected in a small subset of invasive vulvar cancer cells.