p27 and cyclin D1 abnormalities in uterine papillary serous carcinoma.

OBJECTIVE: The expression status of p27 and cyclin D1 was examined in 21 uterine papillary serous carcinoma (UPSC) specimens to determine the role of these genes in the development of this disease. The status of p53, p16, Rb, and K-ras was also determined in these tissues so that a marker profile for UPSC could be compared with the published marker profile for other forms of endometrial and ovarian cancer. METHODS: Immunohistochemistry was performed on 21 UPSC tissue sections to determine the expression status of p27, cyclin D1, p53, p16, and Rb. K-ras mutations were identified by restriction fragment length polymorphism analysis of DNA isolated from the UPSC sections. RESULTS: All specimens displayed at least one molecular abnormality. A high incidence of p27 alterations were observed, with reduced p27 expression measured in 16 of 21 (76%) tumors, followed by p53 alterations observed in 13 of 21 (62%) tumors. The p27 abnormalities occur at an early stage of the disease, with 63% (5/8) of Stage I cases displaying reduced p27 expression. Cyclin D1 overexpression was observed in 4 of 21 (19%) specimens, whereas p16, Rb, and K-ras abnormalities were each observed in 2 of 21 specimens (10%). Both K-ras mutations were at codon 12. The p16 and Rb abnormalities coexisted in the same specimens. CONCLUSION: UPSC tumors display a high incidence of p27 abnormalities, suggesting that p27 abnormalities play an important role in the development of this disease. Our results also indicate that cyclin D1 overexpression is involved in the development of a small number of UPSC cases. A comparison of our results with reports by other authors suggests that UPSC shares molecular marker alterations with both ovarian cancer and endometrioid adenocarcinoma.

cJun overexpression in MCF-7 breast cancer cells produces a tumorigenic, invasive and hormone resistant phenotype.

We have previously demonstrated decreased Jun/AP-1 activity in the breast cancer cell line MCF-7 when compared to normal or immortalized mammary epithelial cells. In this paper, we overexpress Jun in MCF-7 cells (MCF7Jun) and demonstrate that it results in diverse biologic and biochemical changes, which mimic those seen clinically in breast cancer. Overexpression of Jun causes significant alterations in the composition of AP-1, decreased junB and increased fra-1 expression and results in an increased biologic aggressiveness. MCF7Jun cells exhibit increased cellular motility, increased expression of a matrix degrading enzyme MMP-9, increased in vitro chemoinvasion and tumor formation in nude mice in the absence of exogenous estrogens. Furthermore, MCF7Jun cells are unresponsive to the growth stimulating effects of estrogen and growth inhibitory effects of tamoxifen. Analysis of the estrogen receptor (ER) expression and activity showed that the MCF7Jun cells have no detectable ER. MCF-7 cells overexpressing mutant forms of cJun were responsive to the growth stimulatory effects of estrogen indicating that full-length cJun is required to acquire the estrogen-independent phenotype in breast cancer cells.

Retinoic acid receptor beta expression and growth inhibition of gynecologic cancer cells by the synthetic retinoid N-(4-hydroxyphenyl) retinamide.

BACKGROUND: The synthetic retinoid N-(4-hydroxyphenyl) retinamide (4HPR) can inhibit the growth of tumor cells. Preliminary results from a clinical trial suggest that 4HPR may reduce ovarian cancer incidence. We examined the growth-inhibitory effects of 4HPR on gynecologic cancer cell lines in vitro and the role of retinoid receptors in modulating this effect. METHODS: Twelve human gynecologic cancer cell lines (the ovarian cell lines--A224, AD10, UCI 101, UCI 107, SKOV3, 222, CP70, ML3B, and ML5; the cervical cell lines--HT3 and ME180; and the endometrial cell line--Hec 1A were tested for sensitivity to 4HPR (by assaying cell proliferation rates). Gel electrophoretic analysis of DNA fragmentation was used to measure programmed cell death (apoptosis). Specific retinoid receptor (retinoic acid receptor [RAR] and retinoid X receptor) messenger RNA (mRNA) levels were measured by northern blot hybridization. AD10 cells were stably transfected with human RARbeta complementary DNA, and the effect of 4HPR on cell proliferation was examined. RESULTS: 4HPR inhibited the growth of all 12 cell lines, but to varying degrees; IC50 values (i.e., concentrations that inhibit proliferation by 50%) ranged from 0.3 to 9 microM. Following 4HPR treatment, ovarian cancer cells that were sensitive to 4HPR (222, CP70, and UCI 101; IC50 <3 microM) contained higher levels of RARbeta transcripts than more resistant cells (AD10, ME180, Hec 1A, and A224; IC50 > or =3 microM) (2.8-fold; two-sided P = .006). Anchorage-independent growth of transfected AD10 cells expressing high levels of RARbeta was totally abolished, even in the absence of 4HPR; transfectants expressing low levels of RARbeta exhibited lower levels of anchorage-independent growth and grew more slowly in the presence of 4HPR than control untransfected AD10 cells. CONCLUSION: 4HPR inhibited the proliferation of ovarian cancer cells in vitro; RARbeta expression appeared to be associated with this effect.

FHIT gene expression in human ovarian, endometrial, and cervical cancer cell lines.

The fragile histidine triad (FHIT) gene, located at 3p14.2, has been shown to be altered in numerous epithelial cancers. Because previous studies have shown a loss of heterozygosity and cytogenetic abnormalities at the 3p region in ovarian, endometrial, and cervical carcinomas, we examined the status of the FHIT gene in 14 ovarian, 8 cervical, and 4 endometrial human cancer cell lines. RNA was isolated and subjected to reverse transcription-PCR to amplify the FHIT gene transcript. Sixty-three % (5 of 8) of cervical cell lines, 14% (2 of 14) of ovarian cell lines, and none (0 of 4) of the endometrial cell lines displayed aberrantly migrating FHIT transcripts. DNA sequencing demonstrated that the aberrantly migrating bands primarily lacked exons 5, 6, and 7 (with other exon losses also observed), resulting in shorter mRNA transcripts. Southern blot analysis of DNA from five of the cervical carcinomas demonstrated alterations in four of them, three of which had exhibited no normally sized FHIT transcripts. The results suggest that the expression of the FHIT gene may be altered in cervical tumor tissue, potentially implicating this gene in cervical tumorigenesis, whereas the involvement of this gene appears to be less important in the development of ovarian and endometrial cancer.

Demonstration of a 60K molecular weight luteinizing hormone-releasing hormone receptor in solubilized adrenal membranes by a ligand-immunoblotting technique.

A new technique for the identification of LHRH receptors has been developed and applied to demonstrate an adrenal LHRH binding protein. Solubilized membrane proteins were separated electrophoretically and transferred to nitrocellulose paper. This was followed by sequential incubations with LHRH, anti-LHRH antiserum, peroxidase-conjugated second antibody, and 4-chloro-1-naphthol. Using an antiserum directed towards the middle region of LHRH, a 60K mol wt band was visualized in rat adrenal and pituitary membranes. A band of slightly higher molecular weight was present in membranes of bovine adrenal cortex but was absent in the medulla. The 60K band was not visualized when nonimmune rabbit serum was used. The 60K band was also not visualized when an antiserum requiring the NH2 and COOH termini of LHRH was used, suggesting that these regions of LHRH are not accessible to the antiserum after binding to the receptor. These studies have demonstrated the existence of LHRH binding protein in adrenal cortical tissue with a molecular size similar to that of the pituitary receptor. Adrenal membrane binding sites were less clearly demonstrated by conventional 125I-ligand binding techniques as nonspecific binding was high. The ligand-immunoblotting technique is a sensitive, specific and rapid procedure with potential application in screening normal and tumor tissues for LHRH receptors and studying LHRH interactions with its receptor.