Expression of ras and c-myc oncoproteins and hepatitis B surface antigen in human liver disease.

One of the major antecedent factors preceding the development of hepatocellular carcinoma is chronic hepatitis B virus infection. Also, recent molecular studies have shown that activation of c-oncogenes might be responsible for the malignant transformation in some cases of hepatocellular carcinoma. We used immunohistochemical methods to investigate the correlation of ras and c-myc oncogene expression with the presence of HBsAg in human liver disease. Our material consisted of 23 chronic active hepatitis B needle liver biopsies and surgical specimens from 11 cases of cirrhosis, 23 hepatocellular carcinoma and 10 normal adult livers. Direct, three-step and streptavidin-biotin-complex immunoperoxidase techniques using polyclonal (anti-HBsAg) and monoclonal antibodies (anti-ras p21, anti-myc p62), were performed. Normal liver tissues were negative for all antibodies used. In HBsAg+ chronic active hepatitis B cases enhancement of c-myc, and less frequently of ras oncogene expression, was a common observation. Increased myc p62 and ras p21 expression was a finding not restricted to HBsAg+hepatocytes, which occasionally were negative for oncoprotein immunostaining. All HBsAg-chronic active hepatitis B cases were negative for ras p21 and myc p62 specific staining. Cirrhotic livers showed more frequently enhanced c-myc expression. Most of the immunostained cells were negative for HBsAg. HBsAg- cases of hepatocellular carcinoma more often showed ras p21 than myc p62 overexpression. HBsAg+ hepatocellular carcinomas presented only ras p21-positive immunostaining, which was not detected in HBsAg+ hepatocytes. Our recent data supports the view that continued expression of HBsAg in human liver disease is not necessary for the enhancement of ras and c-myc oncogene expression.

C-erbB-2 expression in squamous cell carcinoma of the head and neck.

Seventy-five squamous cell carcinomas of the head and neck were analysed for c-erbB-2 expression using immunohistochemical techniques with four different c-erbB-2 antibodies. No membrane staining was seen in any of the squamous cell carcinomas studied with any of the antibodies; however, c-erbB-2 cytoplasmic staining was seen in 60 per cent of the tumours. The significance of cytoplasmic staining is discussed and that it may possibly represent elevated c-erbB-2 expression in squamous cell carcinomas. C-erbB-2 cytoplasmic staining was also observed in 10 of 23 normal specimens obtained from the resection margin of the tumours. No correlations were found between positive c-erbB-2 cytoplasmic staining and any of the clinicopathological parameters or survival.

Low levels of ras p21 oncogene expression correlates with clinical outcome in head and neck squamous cell carcinoma.

We have previously demonstrated that the Ha-ras and the Ki-ras oncogenes are overexpressed in squamous cell carcinoma of the head and neck. In this study we have used the Y13-259 monoclonal antibody to p21 ras to determine if expression of the ras oncoprotein correlates with any of the clinico-pathological parameters or with survival in 69 patients with squamous cell carcinoma of the head and neck. Forty-four specimens were from patients with previously untreated tumours and 25 from patients with previously treated disease. We have found a correlation between low levels of ras expression and the disease-free survival period in patients with previously untreated tumours. Three per cent of the patients with ras negative staining were alive 60 months after diagnosis, whereas 54 per cent of the patients with positive staining were still alive after the same time period (P less than 0.05).

Elevated P53 expression correlates with a history of heavy smoking in squamous cell carcinoma of the head and neck.

Expression of the tumour suppressor gene p53 was examined in squamous cell carcinoma of the head and neck using two p53 antibodies, PAb 421 and PAb 1801. Elevated p53 expression was found in 67% of the 73 patients investigated. P53 expression was not found to correlate with whether the patient had been previously treated or not, nor any of the clinico-pathological parameters. However a correlation was found between the patients smoking history and positive p53 staining. Six out of seven non-smokers did not express p53 whereas 29 of 37 heavy smokers were found to have elevated p53 expression (P less than 0.005). Also, of a group of ten patients who had given up smoking more than 5 years ago, nine had elevated expression. Epidemiological studies have shown a correlation between heavy smoking and head and neck cancer. The present study indicate a genetic link for this correlation.

Immunohistochemical analysis of the expression of the c-myc oncoprotein in human stomach cancers.

In an immunohistopathological study, we have used the specific monoclonal antibody myc 1-9E10 to the c-myc oncoprotein in 88 gastric carcinomas (22 gastric biopsies and 66 gastrectomies for cancer). Positive myc p62 immunoreactivity was shown in 48 (55%) cases with moderate or intense staining. The remaining 40 cases exhibited negative or equivocal staining. Normal stomach mucosa was generally nonreactive, with the exception of parietal cells. Elevated c-myc expression was not found to correlate with histological differentiation or in patients with metastases in one or more perigastric lymph nodes. A correlation was found between the level of c-myc expression and the stage of the disease, (p = 0.04); positive c-myc staining was found in 0/4 early gastric cancers and in 48/84 with advanced disease. Also, an association was found between the elevated c-myc expression and depth of invasion (p = 0.1; 0/4 mucosa and submucosa, 2/6 muscularis propria and 25/47 serosa). The c-myc monoclonal myc 1-9E10 may therefore be of use as a marker of advanced disease and depth of invasion in stomach cancer.

Ras, C-myc and C-erbB-2 oncoprotein expression in non-AIDS Mediterranean Kaposi's sarcoma.

We have studied ras p21, c-myc p62 and c-erbB-2 oncogene expression in fourteen Greek patients with NON AIDS Mediterranean Kaposi's sarcoma using immunohistochemical analysis. Elevated expression of ras p21 expression was observed in all 14 cases studied (8 of which had intense levels of staining), whereas expression of c-myc and c-erbB-2 was less frequent, (6 out of 13 cases tested showed elevated c-myc expression and 6 out of 13 showed elevated c-erbB-2 expression). This report indicates that aberrant ras p21 oncogene expression is a feature of Kaposi's sarcoma and may be important in the early stages of this disease.

Evaluation of the ras and myc oncoproteins in benign gastric lesions.

Ras and c-myc oncoprotein expression was analyzed using specific monoclonal antibodies Y13-259 (for ras p21) and mycl-9E10 (for c-myc p62) in 144 histological sections derived from benign gastric lesions. Increased expression of ras p21 was observed in inflammatory metaplastic, dysplastic, hyperplastic and cystic histological changes, and on the basis of ras p21 staining three distinct histological groups emerged: (i) cystic changes, hyperplastic polyps; (ii) inflammatory gastritis; (iii) metaplastic, dysplastic and adenomatous polyps. Elevated levels of ras p21 expression were found at significantly higher levels than those of c-myc expression in dysplastic lesions. However the expression of the c-myc oncoprotein was less frequent than ras p21 in all other histological types. With the exception of parietal cells, normal stomach mucosa was found to express low levels of both ras p21 and c-myc oncoproteins.

Expression of ras p21 and myc p62 oncoproteins in small cell and non small cell carcinoma of the lung.

Ras p21 and myc p62 expression has been examined immunohistochemically in seventy specimens of bronchial carcinomas. Both ras and myc oncoproteins were found to be overexpressed at a higher frequency in non small cell carcinomas (squamous cell carcinomas and adenocarcinomas) compared to the small cell carcinoma specimens; however only myc p62 overexpression was found to be statistically significant. Also, ras p21 oncoprotein expression was frequently overexpressed in adenocarcinomas compared to squamous cell carcinomas (p less than 0.05). Overexpression of c-myc p62 was found to correlate with poorly differentiated squamous cell carcinomas compared to the well and moderately differentiated tumors. The results of this study indicate that both the ras and myc oncogenes are important in the progression of bronchial carcinomas.

Immunocytochemical study of RAS oncoprotein in cytologic specimens of primary lung tumours.

We have examined the distribution of ras p21 oncoprotein expression in cytologic specimens from 73 primary bronchial carcinomas using an immunocytochemical analysis. The cytologic preparations studied represent the two major groups of histological types of lung cancer: Small Cell Lung Carcinoma (SCLC) and Non-Small Cell Lung Carcinoma (NSCLC) (squamous cell carcinoma and adenocarcinoma). The differential expression of ras p21 oncoprotein correlated with histological classification and was found in 30% of 23 small cell lesions, 61% of 28 squamous cell lung carcinomas and 32% of 22 adenocarcinomas. The ras p21 oncoprotein was commonly expressed in NSCLC cases (48%) as compared to SCLC cases (30%).

Expression of the ras oncoprotein in gastric carcinomas and adjacent mucosa.

We have employed an immunohistochemical analysis to study the ras p21 oncoprotein in a total of 88 gastric carcinomas, which were associated (in 24 cases) with intestinal metaplasia. Our results suggest an association of the expression of ras p21 with metaplastic and neoplastic gastric mucosa. The comparative study showed that 58 of the 88 gastric carcinoma cases studied exhibited negative or equivocal staining (-/+). The remaining 30 were positive with moderate (+) or intense (++) staining. There was an agreement in that histologic type and tumor grade had a strict correlation with staining intensity. Intestinal metaplasia had a higher percentage of positively stained cells (+ or ++). Moreover, there was a selective positive staining in the parietal cells of the gastric fundus in sections from adjacent non-neoplastic mucosa.

ras, c-myc and c-erbB-2 oncoproteins in human breast cancer.

The expression of ras, c-myc and c-erbB-2 oncoproteins in 100 human (73 ductal and 27 lobular) breast carcinomas has been examined using an immunohistochemical analysis. The monoclonal antibody Y13 259 has been used for the ras p21, the monoclonal antibody Myc1-9E10 for the c-myc p62 and the polyclonal antibody pAb1 (from Triton Bioscience Inc.) for the c-erbB-2 p185 oncoproteins. The following conclusions can be drawn from the analysis: Of the 100 breast carcinoma cases studied only 14 did not express any of the three oncogenes. The remaining 86 were positive for one or more of the three oncoproteins. Ductal carcinomas expressed oncoproteins in 92% of the cases (67/73), whereas lobular carcinomas expressed them in 70% of the cases (19/27). The most frequently expressed was c-myc p62 in 70% of cases followed by ras p21, 55% and c-erbB-2, 35%. Elevated expression of ras, myc or erbB-2 oncogenes did not correlate with the presence of metastasis in auxiliary lymph nodes, the numbers of infiltrated lymph nodes the grade of the tumor or hormone status. However, there appears to be a correlation between increased ras staining intensity and patient's age, below 50 years.

Human immunodeficiency virus long terminal repeat responds to transformation by the mutant T24 H-ras1 oncogene and it contains multiple AP-1 binding TPA-inducible consensus sequence elements.

We have employed a short term transfection assay to examine the response of HIV-1 LTR to transformation by the human normal and mutant T24 H-ras1 genes. The plasmid pBC12HIVCAT which carries the HIV-1 LTR sequences linked to the reporter gene chloramphenicol acetyl transferase (CAT) was transfected into rat 208F fibroblasts and their derivatives RFHO6N1-1 and RFHO6T1-1 transfectants. RFHO6N1-1 and RFHO6T1-1 express an exogenous human normal or mutant T24 H-ras1 gene respectively. Expression of the mutant T24 but not the normal H-ras1 gene resulted in increased levels of HIV-1 LTR driven CAT activity. We have noted four motifs in the HIV-1 LTR region which resemble TPA-inducible and AP-1 binding consensus sequences. Since H-ras1 fos and jun/AP-1 respond to TPA and T24 H-ras1 is known to induce both fos and jun/AP-1 nuclear transcriptional factors, it is possible that the latter genes play a role in HIV-1 transcription. Moreover, H-ras1 oncogene activation may play an important role in HIV gene expression and in the activation of latent HIV.

Immunohistochemical analysis of the ras oncogene protein product in human thyroid neoplasms.

The monoclonal antibody Y13 259 to the ras oncogene p21 product was used in an immunohistochemical study of ras expression in human thyroid neoplasms. Both papillary and follicular carcinomas showed consistently higher staining intensity than normal tissue adjacent to carcinomas. Some follicular, foetal, embryonal and oxyphil adenomas exhibited higher staining intensity than normal tissue. Our results suggest that elevated ras expression may be important in the development of some adenomas and in their conversion to carcinomas.

Interleukin 3 like activity secreted from human ras or myc transfected rodent cells.

We have examined the secretion of IL-3 like activity from recipient normal mouse, rat and Chinese or Syrian hamster cells and their derivative cell lines obtained after transfection with recombinant plasmids carrying an exogenous human ras or myc gene. IL-3 like activity was determined by two cell proliferation assays which employed the FDC-P1 and 32D IL-3 dependent mouse cell lines. The colorimetric MTT assay, where a tetrazolium salt is employed, and the trypan blue dye exclusion assay were used. Low serum containing supernatants from rat or Syrian hamster lines expressing an exogenous human normal or mutant T24 H-ras1 gene supported the proliferation of the IL-3 dependent FDC-P1 or 32D cells. However, supernatants from mouse BalbC or Chinese hamster cells expressing the normal or mutant T24 H-ras1 gene failed to support the proliferation of these cells. In addition, supernatants from rat 208F or BalbC cells transfected with recombinant plasmids carrying a human myc gene supported the proliferation of the two IL-3 dependent cell lines. Our results suggest that introduction of the human Ha-ras1 or human myc gene can trigger the secretion of IL-3 like growth factors in some but not all rodent cells.

Elevated expression of the myc gene in human benign and malignant breast lesions compared to normal tissue.

Expression of the c-myc gene in human breast lesions and in adjacent normal tissue was studied by immunohistochemical analysis. The previously described monoclonal antibody Myc1-9E10 (1) which recognizes the p62 c-myc protein was used in paraffin tissue sections. A total of 101 cases of breast disease examined included 38 simple and complex cystic disease, 18 simple and hyperplastic fibroadenomas, 36 ductal and lobular carcinomas and 9 in situ carcinomas. Whereas the adjacent normal tissue was slightly positive, 25 out of 38 cystic disease, 7 out of 18 fibroadenoma, 36 out of 36 carcinoma and 9 out of 9 in situ carcinoma specimens showed moderate to high levels of p62 c-myc expression as indicated by staining intensity. These results suggest that the c-myc protein may play a role in breast neoplasia.