Expression of ras oncogene p21 protein in relation to regional spread of human breast carcinomas

The oncogenes most frequently detected in human tumors belong to the ras gene family (Ha-ras, Ki-ras, and N-ras). These genes encode a group of closely related 21,000 dalton proteins termed p21. An immunohistochemical study of ras p21 expression was carried out on paraffin sections of 54 human breast carcinomas using monoclonal antibodies to p21. The control group consisted of ten cases of benign fibrocystic disease. The p21 expression was significantly higher in cancer cells than in epithelial cells of control specimens. No correlations, however, were observed between oncogene product expression and tumor size, histologic type, or grade. As a group, tumors with axillary lymph node metastases expressed higher levels of ras p21 than nonmetastasizing tumors. However, because of the significant overlap in individual p21 values, it is unlikely that the immunohistochemical assay for p21 could be used to predict the behavior of mammary carcinomas.     

Expression of the 21,000 molecular weight ras protein in a spectrum of benign and malignant human mammary tissues

Monoclonal antibodies RAP-5 and Y13-259, directed against the ras gene product [a protein with a molecular weight of 21,000 (p21)] have been used to evaluate ras p21 expression in malignant and benign mammary tissues as well as in the lesions of intermediate stature such as atypical hyperplasia using immunohistochemical assays. Invasive carcinoma demonstrated enhanced expression of ras p21, with generally decreasing expression in carcinoma in situ, atypical hyperplasia, and nonatypical hyperplasia, respectively. Heterogeneous expression of ras p21 was observed among primary as well as metastatic mammary carcinomas. Carcinomas from postmenopausal patients generally demonstrated higher levels of ras p21 than those from premenopausal patients, but no significant difference in ras p21 expression in carcinomas between estrogen-receptor rich and estrogen-receptor poor patients was found. Normal mammary epithelium in terminal duct lobular units from patients with hyperplasia generally demonstrated higher levels of ras p21 expression than did epithelium in large ducts. This demonstration of enhanced ras p21 expression by the epithelium of peripheral lobular portion of the breast is consistent with the previous hypothesis that these areas preferentially undergo malignant transformation. Analyses of the limited number of specimens available from patients with 15-yr follow-up revealed a generally higher level of ras p21 in hyperplasia from patients who subsequently developed carcinoma, as compared to those from patients without carcinoma development. However, no conclusions regarding the potential for malignant transformation could be drawn for any individual patient on the basis of ras p21 expression. Concomitant analyses of ras p21 expression in mammary carcinomas and benign lesions using liquid competition radioimmunoassay and immunohistochemical assay demonstrated the complementary nature of these alternative approaches. These results suggest that enhanced ras p21 expression may be involved in the early stages of mammary carcinogenesis but is probably not involved in the maintenance of the transformed phenotype.     

Immunohistochemical study of the ras oncogene expression in human breast lesions

An immunohistochemical study of ras oncogene expression in human breast lesions was carried out using a monoclonal antibody, Y13 259, to the ras encoded p21 protein. A total of 75 cases of breast disease examined included: 33 simple and complex cystic disease; 22 simple and hyperplastic fibroadenomas; 18 ductal, lobular and mixed carcinomas and 2 in situ carcinomas. Most of the complex cystic disease, hyperplastic fibroadenomas and all types of carcinomas showed high p21 expression as indicated by staining intensity. These results suggest that elevated ras expression may play an important role in the development of some premalignant and malignant breast lesions.     

Immunohistochemical study of oncogene product ras p21, c-myc and growth factor EGF in breast carcinomas.

The expression of the oncogene products ras p21, c-myc and the growth factor EGF (epidermal growth factor) was studied immunohistochemically in the tissue of 119 benign and malignant human breasts. In most cases, histologically normal breast tissues and benign lesions were found to be negative or poorly-expressive for reactivity with each antibody. Similar findings were observed in carcinoma in situ. Invading breast carcinomas demonstrated a significantly higher percentage of stained cells than that observed in benign lesions or carcinoma in situ; forty-two of 66 invasive breast carcinomas (63.6%) were highly-expressive for ras p21, thirty-eight (57.6%) for c-myc and twenty (30.3%) for EGF, but overall correlations between each oncogene expression and the clinical stage, tumor size or degree of differentiation were not found. The overall 5-year survival rate was studied in 58 patients with Stage II and III in association with each oncogene or EGF expression. Their survival rate was significantly effected by the EGF expression (0.05 less than p less than 0.1) but not by ras p21 or c-myc expression. Analysis of 36 specimens available with ER (estrogen-receptor) level revealed a significant correlation between the ER status and c-myc or E2 (estradiol) and a significant inverse correlation between ER status and ras p21 or EGF expression (P less than 0.05). The expression of ras p21, EGF and c-myc was not associated with metastatic tumor progression.     

Evidence of enhancement of the ras oncogene protein product (p21) in a spectrum of human tumors

Using a direct binding liquid competition radioimmunoassay, the amount of the ras oncogene protein product, p21, was quantitated in a variety of human tumors and adjacent apparently normal tissues. In 48 of 50 matched tumor and normal tissue biopsy specimens from 50 patients, more ras p21 was detected in the tumor than in its normal counterpart. Twenty-five of 28 breast tumors demonstrated more ras p21 than the average of the values obtained for fibroadenomas. Furthermore, in 17 of the 19 cases studied, over 20% more ras p21 was observed in breast carcinomas compared with their respective normal counterparts. More ras p21 was also demonstrated in the majority of tumors of the stomach, lung, colon and bladder compared with their respective adjacent normal tissues. Our data therefore indicate that ras p21 expression is quantitatively enhanced in many human tumors originating from several different tissue types.     

c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy

Spontaneously immortalized human skin keratinocytes (HaCaT) were transfected with the c-Ha-ras (EJ) oncogene via a plasmid construct which also contained the selectable neomycin gene. Clones were selected on the basis of G418 resistance. Those clones that had stable integrants of Ha-ras fell into 3 classes with respect to tumorigenicity. Class I clones were nontumorigenic, i.e., formed nodules which rapidly regressed. This phenotype is identical to that seen with parental HaCaT cells. Class II clones formed slowly growing, highly differentiated cystic or papillomatous-type benign tumors, and class III clones formed highly differentiated, locally invasive squamous cell carcinomas. The clones of all three classes exhibited similar morphology and growth potential in culture and retained the ability to reconstitute an epidermis-like stratified epithelium in transplantation experiments. Only the malignant clones showed locally invasive growth. Both the benign and the malignant clones exhibited higher levels of ras integration and variable levels of mutated p21 protein product. Thus, expression of the cellular Ha-ras oncogene in these human epithelial cells significantly altered growth regulation, resulting in varying degrees of growth potential in vivo, ranging from benign to malignant tumors. However, no direct correlation was seen between high levels of p21 expression and malignant growth.     

Development of quantitative liquid competition radioimmunoassays for the ras oncogene and proto-oncogene p21 products

The ras gene family of rodents and humans is highly conserved and consists of several distinct genes, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. This gene family mediates transformation via (1) a point-mutation resulting in the change of one amino acid in the 21 kDA ras gene product (p21) or (2) increased expression of ras p21. Group-specific, type-selective and interspecies indirect binding liquid competition radioimmunoassays (RIAs), capable of providing truly quantitative analyses of the 21 ras oncogene and proto-oncogene products, have been developed. Using purified recombinant ras p21 from Escherichia coli expressing the full-length T24 mutant human Harvey-ras gene protein product as a standard in these RIAs, we have defined the absolute numbers of pg, fM and molecules of ras p21 in: (1) E. coli expressing the point-mutated or proto-ras p21 and (2) mammalian cell lines of human and murine origin. Two of the RIAs developed can be termed group-specific in that they have the ability to detect the point-mutated and proto forms of all 3 human ras genes (Harvey, Kirsten, and neuroblastoma), while the third RIA is type-selective, since it detects an antigenic determinant located primarily on the Harvey ras p21. All 3 RIAs are interspecies-specific since they are able to detect ras p21 in rodent as well as human cells. The adaptability of the RIAs to various assay conditions and ease of methodology make these immunoassays applicable to the study of several parameters associated with ras p21 expression. These assays, used in conjunction with specific cDNA probes to identify specific ras proto-oncogenes or point-mutated oncogenes being expressed, now provide truly quantitative analysis of ras p21 in mammalian cells to further the study of the association between ras p21 expression and transformation.     

ras oncogene p21 as a tumor marker in the cytodiagnosis of gastric and colonic carcinomas

This study was undertaken to determine whether the expression of ras oncogene product p21 can be used as a tumor cell marker of gastric and colonic carcinoma in brush smears. To detect p21 an immunocytochemical assay with RAP-5 monoclonal antibody was used. Benign epithelial gastric cells obtained from normal gastric mucosa or benign gastric lesions reacted negatively in 12 out of 13 cases. Similarly, benign epithelial colonic cells from normal colon or benign colonic lesions were negative for p21 in nine out of ten cases. Weakly positive reaction, confined to a few cell clusters only, was observed in one smear of a benign gastric ulcer and one smear of chronic ulcerative colitis. All 20 smears from colonic carcinoma and all 20 smears of gastric carcinoma contained cells that stained positively for p21, and the degree of tumor differentiation had no impact on the staining pattern. The results recorded in this study show that the immunocytochemical assay for the ras oncogene product may prove to represent a new tool for the cytodiagnosis of gastric and colonic carcinomas.     

Differential expression of p21ras gene products among histological subtypes of fresh primary human lung tumors

Activation of the ras oncogene has been associated with a number of human tumors. In this study, expression of p21ras in different histological types of fresh primary bronchogenic carcinomas was examined. p21ras products were detected in all lung tissues that were analyzed. Only 1 of 23 tumors demonstrated aberrant migration of p21ras. In contrast, 10 tumors had substantially elevated levels of p21ras products with respect to the adjacent normal lung tissues and with respect to the other lung tumors. There was no correlation between increased ras protein expression and tobacco exposure of the patient or extent of disease at the time of diagnosis. However, 9 of 11 tumors with a squamous component as opposed to only 1 of 12 tumors belonging to other histological classifications demonstrated increased p21ras. These data suggest that, in bronchogenic carcinomas, mutations associated with structural abnormalities and aberrant migration of p21ras occur infrequently as compared to quantitative changes in p21ras. Furthermore, differential expression of c-ras products in primary human lung tumors correlates with pathological cell type, and may be a common event in squamous cell carcinomas, but not adenocarcinomas or small cell carcinomas of the lung.     

Expression of p21 ras oncoproteins in human cancers

The expression of proteins encoded by ras oncogenes was examined in 52 fresh human tumors of 19 different types using antibodies generated to different peptide domains of ras proteins of molecular weight 21,000 (p21). Proteins related to ras genes were detected in 90% of tested tumors. In 21% of tumors there were high levels of ras p21 of greater than 40% of the level in a Harvey murine sarcoma virus transformed cell line. Predominance of either cellular Ha-ras or other ras p21s was found in 20 and 14% of tumors, respectively, and predominance of p21 other than Ha-ras was frequent in breast cancers. Abnormal electrophoretic mobility of p21 was observed in two cancers, and a novel rapidly migrating ras p21 was found in a rare malignant fibrohistiocytoma.     

Elevated ras oncogene expression correlates with lymph node metastases in breast cancer patients

The protein product of the ras cellular oncogene(s) (p21) was assayed in primary breast carcinomas from two groups of patients who had different axillary lymph node status. Using an immunohistochemical assay, the intensity and percent of neoplastic cells demonstrating ras p21 antigen staining were significantly higher in the primary tumors from patients with lymph nodes positive (LN+) for malignancy (20 patients) compared with the lymph node negative (LNO) group (21 patients). The expression of p21 also correlated with tumor size. Age and estrogen receptor status did not influence p21 staining. The antigen expression of p21 was similar in intensity and distribution in the primary tumor and regional lymph node metastases. Enhanced expression of p21 in primary breast cancers that metastasize to regional nodes indicates that ras p21 may be a determinant of the malignant potential of breast cancer cells and may represent a new class of more biologically relevant tumor markers.     

Expression of ras oncogene mRNA and protein in aberrant crypt foci.

The expression of the ras oncogene in aberrant crypt foci was studied by both in situ hybridization and immunohistochemical approaches. Aberrant crypt foci are hypothesized to represent the earliest identifiable microscopic lesions of colon cancer in rodent colons. Sprague-Dawley male rats were injected with azoxymethane (20 mg/kg s.c.) once. Twelve weeks later, aberrant crypt foci were identified topographically, microdissected and processed for histology. In situ hybridization with an antisense oligomer of c-ras demonstrated increased expression of ras-specific RNA in aberrant crypts compared to normal crypts. A low amount of non-specific hybridization was obtained with the corresponding sense oligomer. The percentage of cells with grains (labeling index) was calculated in early and advanced aberrant crypt foci. This index was also calculated in normal appearing crypts. The labeling indices for the early and advanced aberrant crypt foci were significantly greater than that of normal crypts (18.0 and 25.0 versus 11.9). In the same tissue specimens, immunohistochemical staining for ras p21 with the monoclonal antibody (Y13-259) revealed strong staining intensity in early aberrant crypts (15/22) and advanced aberrant crypts (22/30) compared to normal crypts (3/50). The immunohistochemical results demonstrate the presence of elevated levels of ras p21 in the same tissue as increased levels of ras-specific message. This investigation provides the earliest demonstration of increased expression of the ras oncogene in precursor lesions of colon cancer possessing dysplastic features.     

Expression of ras oncogene p21 in relation to prognostic factors of human breast cancer]

For the purpose of demonstrating the relationship between the expression of ras oncogene p21 protein and clinico-pathological characteristics which reflected the prognosis, 253 women with breast cancer who underwent mastectomy were analyzed. Ras p21 was detected in 133 (52.6%). In histological types, scirrhous carcinomas were more often ras p21-positive, and papillo-tubular carcinoma were usually negative. And histological grade was significantly correlated with ras p21. The degrees of invasion to fat tissues and infiltration into lymphatic vessels were also significantly correlated with ras p21. Tumors with lymph node metastases expressed higher levels of ras p21 than nonmetastasizing tumors in smaller tumors, especially in papillo-tubular carcinomas. And patients with elevated ras expression tended to have a poor prognosis. These results suggested that an elevated ras expression may play an important role in the development of aggressive tumors.     

Clinical and histopathologic evaluation of the expression of Ha-ras and fes oncogene products in lung cancer.

The expression of Ha-ras and fes oncogenes was investigated with the immunohistochemical method in formalin-fixed, paraffin-embedded tissue specimens of 147 lung carcinomas. Positive immunoperoxidase reactions for Ha-ras p21 were found in 80.5% of the adenocarcinomas, 39.5% of the squamous cell carcinomas, 21.4% of the large cell carcinomas, and 15.4% of the small cell carcinomas; those for fes P85 were found in 51.2% of the adenocarcinomas, 26.3% of the squamous cell carcinomas, 35.7% of the large cell carcinomas, and 15.4% of the small cell carcinomas. Both Ha-ras p21 and fes P85 were expressed most frequently and most strongly in adenocarcinoma. In addition, adenocarcinoma showed significantly higher incidence of concomitant expression of Ha-ras p21 and fes P85 as compared with other histologic types of lung cancer. Thus, the authors suggest that the cooperative effects of Ha-ras and fes oncogenes are especially important in the carcinogenesis of adenocarcinoma. In adenocarcinoma, the incidence and grade of Ha-ras p21 expression increased with the degree of histologic differentiation, suggesting that Ha-ras oncogene might be related to cellular differentiation. Papillary adenocarcinoma showed more frequent Ha-ras p21 expression in comparison with acinar adenocarcinoma. In well- or moderately differentiated adenocarcinoma, the incidence and grade of Ha-ras p21 immunoreactivity in the cases with poor prognosis were significantly higher than in those with good prognosis if other major prognostic factors were equivalent in the two groups. The authors propose that the expression of Ha-ras p21 may be one of the useful prognostic factors in such carcinomas.     

Activation of the cellular Harvey ras gene in mouse skin tumors initiated with urethane

Mouse skin tumors, benign papillomas, and squamous cell carcinomas (SCCs) were initiated by a single topical application of urethane followed by repeated promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Using the NIH 3T3 focus forming assay, dominant transforming activity was detected in DNA isolated from SCC samples. Rearranged and amplified copies of the c-Ha-ras gene were detected in NIH 3T3 transformant cell lines, indicating that an activated Ha-ras gene had been transferred to the NIH 3T3 recipient cells. Analysis of p21ras from the transformant cell lines suggested that the activating ras mutation was present in codon 61. Ultimately, the Ha-ras gene was shown to be activated by a specific A----T transversion at the second position of codon 61. This mutation was detected in both benign papillomas and SCCs, suggesting the activation occurred early in tumor development. The results demonstrate a highly consistent activation of the Ha-ras oncogene by a specific point mutation, suggesting a functional role for an activated ras gene in the initiation of mouse skin tumors by urethane.     

Protection against malignant conversion in SENCAR mouse skin by all Trans retinoic acid: Inhibition of the ras p21-processing enzyme farnesyltransferase and Ha-ras p21 membrane localization

Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha-ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane-bound Ha-ras p21 were determined in all papillomas and carcinomas obtained from acetone- or RA-treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA-treated groups showed significantly decreased (P < 0.01–0.001) FTase activity. Furthermore, the tissue samples from RA-treated groups in different protocols also showed significantly diminished membrane localization of Ha-ras p21, with a concomitant increase in cytosolic Ha-ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha-ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene. © 1996 Wiley-Liss, Inc.     

Evaluation of a monoclonal antibody to ras peptide, RAP-5, claimed to bind preferentially to cells of infiltrating carcinomas

RAP-5, a monoclonal antibody raised against a p21ras peptide, has been claimed to show immunohistochemical localisation of cells with infiltrative properties in human tumours. We confirmed that this antibody reveals pronounced cellular heterogeneity in human colonic neoplasms but could find no obvious relationship to infiltrative activity. RAP-5 bound to many different cell types, neoplastic and normal. In order to clarify the specificities of RAP-5 we applied it to two cell lines: nontumorigenic hamster fibroblasts in which ras expression is barely detectable, and a vigorously tumorigenic line derived from these fibroblasts by insertion of the human mutated Ha-ras oncogene in a high expression vector. Another antibody to p21ras, Y13-259, clearly distinguished between these cell lines both on immunoblots and immunocytochemically, but RAP-5 did not. Rather, it bound to proteins of a variety of molecular weights in both cell lines. The results show that RAP-5 is unlikely to be a useful reagent for detection of ras associated proteins in human tissues.     

Quantitation of enhanced expression of ras-oncogene product (p21) in childhood renal tumours.

A monoclonal antibody, RAP-5, raised against the 21,000d ras-oncogene product, p21, was used in an immunoperoxidase staining procedure to study the expression of p21 in normal children's and foetal kidneys, pre-malignant lesions and benign and malignant childhood renal tumours with good, moderate or poor prognosis. ras-p21 was expressed in both normal and foetal kidneys but its distribution in renal tumours differed markedly (p less than 0.01). A quantitative liquid competition radioimmunoassay (RIA) was used to determine ras-p21 level in tissue homogenates. The results were expressed as pg of ras-p21 per microgram protein of tissue extract. There were significant differences in the levels of ras-p21 among various renal tissue extracts (p less than 0.05). Generally it emerged that the amount of ras-p21 was greater in both malignant renal tumours and foetal kidneys compared to normal kidneys, pre-malignant lesions and benign renal tumours.     

Immunohistochemical demonstration of ras p21 oncogene product in normal, benign, and malignant human thyroid tissues

The p21 protein product of the cellular oncogene ras, designated ras p21, has been detected immunohistochemically in normal, benign and malignant human thyroid tissues. With the monoclonal antibody RAP-5 generated against a synthetic peptide corresponding to amino acid positions 10 to 17 of the ras p21 protein and an avidin-biotin-peroxidase complex (ABC), the expression of the ras p21 was evaluated in paraffin-embedded sections. Western blot analysis using fresh thyroid carcinoma tissue revealed double protein bands, one band was at molecular weight 21,000 and the other was a more rapidly migrating band at the molecular weight 17,500. Immunohistochemically, papillary adenocarcinomas of the thyroid showed moderate to intense stainings for ras p21 in most cases. Cytoplasmic and apical surface stainings were the most common patterns of immunoreactivity. Adenomas showed variable ras p21 positivity in cytoplasm and apical surface stainings of adenomas were negative to borderline in most cases. The cytoplasm of tissues of Hashimoto's thyroiditis, Graves' disease, and normal thyroid tissues was uniformly ras p21 positive, but the apical cell surface was nonreactive for ras p21 in all tissues. Judging from the findings obtained on this large series of normal, benign, and malignant thyroid tissues, the elevation of ras p21 may be a common event in thyroid neoplasm, and especially elevated ras expression in the apical cell surface may be characteristic to papillary carcinomas of the thyroid. This suggests that apical surface expression of ras p21 may be important in the development of thyroid carcinomas and be useful in differentiation of papillary adenocarcinoma.