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Normal ovarian and ovarian cancer tissues from 35 patients were tested for LDH-A-ras protein complex formation, as well as for the expression of ras and LDH-A genes. By immunodetection of the immunoprecipitates, LDH-A-ras protein complexes are present in the homogenates of human ovarian tissues (benign and malignant). Two bands of 21 and 36 kDa were demonstrated in affinity-purified LDH active fractions by Western blot analysis, and these two proteins were proved to be ras p21 and LDH-A by immunodetection. By Northern blot analysis, elevated levels of c-Ki-ras and LDH-A mRNA transcripts were noted in 49% (17/35) and 40% (14/35) of ovarian cancers, respectively. Concurrent high expression of both genes was demonstrated in 11 cases (31%). Tyrosylphosphorylation of LDH-A was demonstrated in normal and malignant ovarian tissues, and the extent of phosphorylation was correlated with the stage of ovarian cancer. The concurrent elevated expression of ras and LDH-A in the same ovarian tissue may imply that the increased synthesis of LDH-A is due to increased or amplified signaling of the ras-cascade pathway. The possible biochemical role of the tyrosylphosphorylated LDH-A complexed with ras p21 in cell transformation and progression of human ovarian cancer is worthy of further investigation.

Immunohistochemical techniques for the detection of oncogene products and the assessment of cell kinetics can represent promising investigational tools in clinical oncology. In the present paper the immunohistochemical expression of p185, p21 and proliferating cell nuclear antigen (PCNA) was retrospectively assessed in formalin-fixed, paraffin-embedded tissue samples taken from 28 primary ovarian carcinomas at first surgery. Positive immunostaining for p185 was found in 0% of 6 Stage I and 23% of 22 Stage III-IV tumors. Positive immunostaining for p21 was observed in 0% of early and 41% of advanced carcinomas; this immunohistochemical finding correlated significantly with histologic grade (G3 vs G1-2 = 47% vs 9%, p = 0.042). Elevated PCNA immunoreactivity was detected in 33% of Stage I and 50% of Stage III-IV tumors. Among the 20 patients with advanced carcinoma who underwent cisplatin or carboplatin based chemotherapy followed by second-look laparotomy, the pathologic complete response (pCR) rate was 36% for patients with low PCNA expression and 0% for those with elevated PCNA expression. A tendency towards a higher pCR rate was also found for patients with negative immunostaining for p185 or for p21. The prognostic value of the immunohistochemical detection of p185, p21, and PCNA in ovarian carcinoma deserves to be further investigated.

Objective: The aim of the study was to investigate the biologic significance of p21 expression in normal and neoplastic ovarian tissues., Study Design: Western blotting analysis of p21/ras oncoprotein was conducted in a group of 14 normal and cystic ovaries, six benign tumors, 42 primary ovarian cancers, and 15 omental metastases., Results: Levels of p21 were similar in normal and cystic ovaries and in benign tumors, whereas they were significantly higher in malignant tumors than in control tissues (median 1.91, range 0.12 to 5.00 vs median 1.03, range 0.32 to 2.20; p = 0.023) and in omental metastases than in primary ovarian carcinomas (median 3.05, range 0.55 to 5.72 vs median 1.97, range 0.12 to 5.00; p = 0.14). We found no correlation between p21 expression and histopathologic or clinical characteristics. Estrogen receptor-positive and progesterone receptor-positive tumors expressed higher p21 levels than did estrogen receptor-negative and progesterone receptor-negative tumors (p < 0.05), but no correlation with epidermal growth factor receptor status was found. In the univariate analysis of survival p21 positivity showed a negative prognostic value., Conclusion: The enhancement of p21 protein is associated in the ovarian tissue with the malignant phenotype and the acquisition of metastatic potential.

Monoclonal antibody rp-28 directed against the ras gene product p21 has been studied to evaluate ras p21 expression in malignant and benign ovarian tissues. Some ovarian carcinomas of serous and mucinous cystadenocarcinomas, undifferentiated adenocarcinomas, and clear cell carcinomas demonstrated intense staining of ras p21. The frequency and intensity of ras p21 staining were observed to increase with the degree of malignancy. There was no significant difference in ras p21 expression between early and late stages in ovarian tumors arising from the coelomic epithelium. With respect to prognosis, no differences between the ras p21-positive and -negative cases in ovarian tumors arising from the coelomic epithelium were observed. It is, therefore, possible to say that ras p21 expression was not related to clinical staging and prognosis. Expression of ras p21 in malignant lesions was higher than that in benign lesions of the ovary, and the expression is associated with the degree of malignancy in some types of ovarian tumors. Overexpression of ras p21 was observed in epithelial tumors; however, increased expression was not observed in germ cell and sex-cord stromal tumors. This differential expression of ras p21 is due to the different histogenesis of ovarian tumors. This fact may reflect a different carcinogenic mechanism for different types of malignancy.

We have investigated the expression of the products of two well-characterized oncogenes, erbB and Ha-ras, in the primary cultures of human gynecologic tumor cells by indirect immunofluorescent analysis. Epidermal growth factor receptor (c-erbB gene product) and p21 (ras gene product) were highly expressed in 21 out of 26 cases (81%) and 14 out of 18 cases (78%), respectively. Furthermore, they were frequently co-localized in the same region of the cells. These results suggest that these two oncogene products may play a part in gynecologic tumorigenesis and that they can interact with each other.

Flow cytometric analysis of DNA content and ras p21 expression were studied in paraffin-embedded normal ovary (NO, n = 10), serous cystadenoma (SA, n = 11), serous tumors of low malignant potential (LMP, n = 13), and papillary serous cystadenocarcinoma (SCa, n = 7). Tissue for DNA analysis was processed via a modified Hedley technique; a separate aliquot of the same sample was stained with a monoclonal antibody directed to oncoprotein ras p21 (clone Y13259). NO (10/10) and SA (11/11) demonstrated diploid DNA stemlines; 4/12 LMP and 5/7 SCa were aneuploid. S-phase fraction varied with significant differences (p less than 0.001) for SA (mean = 4.4%), LMP (mean = 9.0%), and SCa (mean = 12.7%). When all cases were evaluated, p21 expression was relatively lower in NO and SA (mean = 4.2%) in contrast to LMP and SCa (mean = 13.0% and 8.1%). Diploid cases were examined separately, and lesions with high p21 expression were associated with a diagnosis of LMP/SCa (p less than 0.001), whereas diploid tissues with low p21 expression were associated with a nonmalignant diagnosis (NO/SA). This study suggests that aneuploidy or diploidy with high p21 expression (greater than 10%) may be associated with LMP or frankly malignant ovarian tumors.

In the present study, ras oncogene product p21 was analyzed immunohistochemically in rat ovarian tumors induced by 7,12 dimethylbenz (a) anthracene (DMBA). 1) After 28-38 (average 35.6) weeks, the tumors -6 adenomas, 30 adenocarcinomas, 3 sarcomas, one mixed müllerian tumor and 2 epidermal cysts--were produced in the rat ovaries. 2) The p21 positive reactions could be seen in the cytoplasm of the tumor cells. The positive rates were as follows: adenomas 83%, adenocarcinomas 57%, sarcomas 67%, mixed müllerian tumors 0%, epidermal cysts 100%, in which the positive reactions were obtained in squamous cells. Both of the serial-allografted tumor and DMBA-OC-1 were positive. 3) The present study showed that the ras oncogene is not a specific gene in ovarian sarcoma or in other tumors. 4) The present study has suggested that the ras oncogene plays a role in tumor genesis including benign tumors, by showing p21 positive in adenomas and epidermal cysts.

In the present study, monoclonal antibody rp-28 directed against the ras gene product p21 was used to evaluate ras p21 expression in malignant and benign ovarian tissues. Some ovarian carcinomas (serous cystadenocarcinoma, mucinous cystadenocarcinoma, undifferentiated carcinoma and clear cell carcinoma) demonstrated intense staining of ras p21. In mucinous tumors, both the frequency of ras p21 positive staining and the staining intensity gradually increased with the degree of malignancy. There was no difference in ras p21 expression according to the clinical stages of ovarian carcinomas. In the metastatic lesions, ras p21 staining was rather weaker than in the primary lesions. It is therefore possible that intense staining of ras p21 is associated with the degree of malignancy in some types of ovarian tumors, and that the expression of ras oncogene product p21 is not enhanced with progression and metastasis in such types of ovarian carcinoma. These results suggest that ras oncogene plays an important role in the carcinogenesis of some types of epithelial ovarian tumors.