Inhibition of ras p21 membrane localization and modulation of protein kinase C isozyme expression during regression of chemical carcinogen–induced murine skin tumors by lovastatin

We investigated the ras p21 membrane localization and the expression and activation of protein kinase C (PKC) isozymes in activated ras oncogene–containing tumors and assessed whether these events were related to tumors growth. We used 7,12-dimethylbenz[a]anthracene–initiated and 12-O-tetradecanoylphorbol-13-acetate–promoted SENCAR mouse skin tumors, which were shown to contain Ha-ras oncogene activated by point mutation at codon 61, as an in vivo model for these studies. Compared with levels in epidermis, highly elevated levels of membrane-bound Ha-ras p21 were observed in growing tumors, which also showed strong expression and membrane translocation of PKC ζ and βII and weak expression of PCK α. However, when ras p21 membrane localization was blocked in vivo in growing tumors by lovastatin, opposite results were evident. Compared with saline-treated animals, in which tumor growth continued, lovastatin-treated animals had significantly inhibited tumor growth, which led to tumor regression with concomitant inhibition of Ha-ras p21 membrane localization. These regressing tumors from lovastatin-treated animals also showed a decrease in the expression and membrane translocation of PKC ζ and βII but increased expression of PKC α. Taken together, our results indicate that ras p21 membrane localization and the expression and activation of PKC ζ, βII, and α may be the critical events in the regulation of the growth of tumors that contain activated ras oncogenes. © 1995 Wiley-Liss Inc.     

p53 protein expression and cell proliferation in non-neoplastic and neoplastic proliferative skin diseases

AIMS AND BACKGROUND: The p53 protein is essential for the regulation of cell proliferation and its aberrant accumulation is usually seen in malignant tumors but also occurs in squamous epithelium of inflammatory skin diseases characterized by hyperproliferation. The aim of this study is to elucidate the role of the p53 tumor suppressor protein in the pathogenesis of different hyperproliferative, non-malignant and malignant skin diseases, and to determine the association between p53 overexpression and cell proliferation. We also investigated the influence of aging on p53 and Ki-67 protein expression. METHODS: One hundred and fifty skin specimens divided into 30 samples each of normal skin (NS), psoriatic skin (PS), keratoacanthomas (KA), basal cell carcinomas (BCC), and squamous cell carcinomas (SCC) were examined immunohistochemically to assess p53 and Ki-67 protein expression. RESULTS: p53 immunostaining of NS, PS, KA, BCC and SCC was detected in 39.0%, 46.7%, 66.7%, 80% and 86.7% of cases, respectively. Median values and ranges of p53 protein expression were as follows: 0.0% (range, 0.0-1.8%) in NS, 0.0% (range, 0.0-6.5%) in PS, 9.2% (range, 0.0-24.0%) in KA, 19.3% (range, 0.0-48.1%) in BCC and 30.1% (range, 0.0-68.1%) in SCC. p53- and Ki-67-positive cells were present in basal (NS) and suprabasal layers (PS), and not only in cancer nests of KA, BCC and SCC but also in dysplastic and even morphologically normal epidermis adjoining cancers. The positivity of p53 and Ki-67 proteins differed significantly among the groups, with no differences in p53 expression between NS and PS and in Ki-67 expression between PS and KA. Within all groups there was a significant correlation between p53 and Ki-67 expression. Lesion location and patient age, with the exception of location in PS and age in BCC, were significantly related to p53 and Ki-67 expression in all groups. CONCLUSIONS: Our findings suggest that p53 overexpression occurs mainly in neoplastic skin lesions, although it may also occur in squamous epithelium of inflammatory skin diseases such as PS, as well as in normal skin epithelium. It is associated with cell proliferation in normal as well as altered epithelium. p53 protein overexpression is an age-related process and significantly associated with sun exposure, especially in NS and PS but also in KA and SCC. Our findings suggest that Ki-67 rate and p53 protein expression reflect the degree of malignancy in the examined cutaneous neoplasms.     

Analysis of ras gene expression in stomach cancer by anti-ras p21 monoclonal antibodies

Using anti-ras p21 monoclonal antibodies, RASK-3, which reacts with all of Ki-, N-, and Ha-ras p21, we examined by immunohistochemistry the expression of p21 in human gastric cancer (80 cases) and benign gastric lesions (32 cases). Ten percent formalin fixed tissues were studied. Ras p21 was positive in 51 cases (64%) out of 80 cases and partially positive in 12 cases (15%) at the cancerous areas. Ras p21 was partially positive in 7 cases (9%) at the noncancer areas of the same slides. Intestinal metaplasia and normal parietal cells were also often positive. In the study of 32 cases of benign stomach lesions, 2 out of 3 cases of atypical hyperplasia (ATP) and 3 out of 11 cases of stomach ulcer with regenerating epithelials were positive. Ras p21 was more dominantly expressed in the well-differentiated type of stomach cancer than the poorly differentiated type. Expression of ras p21, however, was not correlated either with the grades of cancer invasion or with the types of cancer infiltration.     

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.     

H-ras activation and ras p21 expression in bladder tumors induced in F344/NCr rats by N-butyl-N-(4-hydroxybutyl)nitrosamine

Bladder tumors were induced in male F344/NCr rats by administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at 500 p.p.m. in their drinking water for 12 weeks. Twenty-one bladder tumors that developed between 25 and 50 weeks after BBN administration was begun were evaluated for immunoreactivity with polyclonal or monoclonal antibodies raised against ras p21, for amplification of ras genes by Southern blotting, and for activating point mutations in ras genes by selective oligonucleotide hybridization of products from polymerase chain reaction (PCR). Increased expression of ras p21 was detected by avidin-biotin immunohistochemistry in 18/21 (85%) of the neoplastic bladder lesions. By Southern analysis, there was no significant amplification of H-ras, K-ras or N-ras in any of the tumors except one that showed a 5-fold amplification of K-ras. Point mutations in ras genes were detected by selective oligonucleotide hybridization of the products of PCR. Of the 21 bladder tumors, three tumors were shown to have mutations in codon 12 (GGA----GAA), six tumors in codon 61 (two CAA----CTA, four CAA----CGA), and one in both codon 12 (GGA----GAA) and codon 61 (CAA----CGA), all in H-ras. Thus 10 of 21 tumors has ras gene mutations in a portion of the tumor cells. The variable pattern of point mutation in H-ras suggests that these mutations may not all be a direct consequence of interaction of BBN metabolites with H-ras. Enhanced expression of ras p21 was always focal and was not necessarily associated with transforming ras mutations. It is therefore suggested that tumorigenesis in BBN-initiated bladder cells might involve H-ras activation as part of a multistep pathway; however, H-ras involvement is not obligatory for tumor development.     

Expression of ras oncogene p21 protein in esophageal squamous cell carcinoma

In order to investigate the value of ras oncogene expression as a prognostic indicator in esophageal squamous cell carcinoma, we evaluated the level of ras oncogene protein product (p21) in 52 specimens resected between 1977 and 1986. All patients were followed until death or for at least 2 years. Pathology slides and archival paraffin blocks were retrieved for confirmation of the original diagnosis, study of histopathologic features, and measurement of p21 content. P21 titers were obtained using the RAP-5 monoclonal antibody in a semiquantitative immunohistochemical assay. Titer was expressed as the highest dilution of antibody giving definitive staining using the avidin-biotin peroxidase method. Ras oncogene was expressed in 88.5% of the specimens. We did not find a significant correlation between ras expression and any of a variety of clinical and histopathologic prognostic parameters. Although patients' median survival after resection of specimens with ras oncogene expression was less than half the median survival after removal of tumors without such expression, this difference was not statistically significant. Further prospective investigations are needed to assess the role of ras oncogene evaluation in clinical practice.     

Inhibition of DMBA/croton oil-induced two-stage mouse skin carcinogenesis by diphenylmethyl selenocyanate.

Selenium, an essential micronutrient, is associated with antioxidant functions, physiological defence mechanisms against different diseases including several types of cancers. Search for new selenium compounds with more chemopreventive activities and lesser toxicities are in progress. In the present study, the antioxidative roles of a synthetic organoselenium compound, diphenylmethyl selenocyanate, were evaluated against 7,12-dimethylbenz(a)anthracene (DMBA)/croton oil-induced two-stage mouse skin carcinogenesis model. The compound was administered orally in carcinogen-induced mice in two different non-toxic doses: 2 mg/kg body weight and 3 mg/kg body weight. Significant inhibition in the incidence of papilloma formation (58-80%) as well as in the cumulative number of papilloma per papilloma-bearing mouse were observed in the treated groups as compared with the carcinogen control group. The compound was also found to significantly upregulate different phase II detoxifying enzymes in liver cytosol such as glutathione-S-transferase (P<0.01), catalase (P<0.01) and superoxide dismutase (SOD) (P<0.01) when measured after 15 days and also after 12 weeks of first DMBA treatment. Lipid peroxidation measured as the thiobarbituric acid reactive substances in liver microsomes was significantly inhibited (P<0.05) in a dose-dependent manner by diphenylmethyl selenocyanate. Thus the compound exerts its chemopreventive activity by reducing papilloma formation during chemically induced carcinogenesis, which in turn, may be through modulating the level of lipid peroxidation and phase II detoxifying enzyme system at the doses evaluated.     

骨肉瘤p21 ras蛋白、p21 wafl/cip1蛋白、PCNA与AgNOR的表达及其临床意义

目的探讨骨肉瘤组织中p21ras蛋白、p21wafl/cip1蛋白、PCNA与AgNOR的表达及其临床意义.方法应用免疫组化方法和组织化学方法检测45例骨肉瘤病人的骨肉瘤组织、20例骨软骨瘤病人的骨软骨瘤组织和20例正常骨组织中p21ras蛋白、p21wafl/cipl蛋白、PCNA与AgNOR的表达情况,对有关临床病理指标综合分析.结果45例骨肉瘤组织中p21ras蛋白、p21wafl/cipl蛋白和PCNA的阳性表达率分别为86.7%、13.3%和100%,AgNOR计数为9.63±2.47,阳性表达率与AgNOR计数均高于骨软骨瘤和正常骨组织;p21ras蛋白的表达与PCNA的表达存在平行关系;AgNOR计数与PCNA的表达呈正相关;四者的表达与骨肉瘤有关临床病理指标之间存在一定的关系.结论检测p21ras蛋白、p21waf1/cip1蛋白、PCNA与AgNOR为骨肉瘤恶性程度和预后的评估提供了重要的辅助参考指标.     

Immunohistochemical study of ras p21 expression in human gastric cancers and benign lesions

Expression of ras p21 in human gastric cancers, benign lesions and normal tissues was immunohistochemically evaluated by the avidin-biotin peroxidase complex (ABC) method with anti-ras p21 monoclonal antibody rp-28. Positive p21 immunoreactivity was shown in 23 (77%) of 30 gastric cancers, in 13 (48%) of 27 benign lesions and in 10 (22%) of 46 normal mucosa cases. Among them, strong staining was demonstrated only in 11 (37%) of 30 gastric cancers, but not in benign lesions and normal tissues. Cases showing more than 20% positive cell ratio were observed in 22 (73%) gastric cancers, in 11 (41%) benign lesions and 6 (13%) normal mucosa cases. Further, intracellular distribution of ras p21 is heterogeneous in gastric cancers, while it is homogeneous in benign lesions or normal tissues. The ABC method with rp-28 could be helpful for clinical differential diagnosis between gastric cancers and benign lesions by investigating three factors: staining intensity, positive cell ratio and intracellular distribution of ras p21.     

PTEN、p21ras在人子宫内膜腺癌组织中的表达及其意义

目的探讨PTEN基因、Ki-ras基因在子宫内膜腺癌中的表达及意义。方法采用免疫组织化学S-P法测定20例正常增生期子宫内膜、20例不典型增生子宫内膜及45例子宫内膜腺癌组织中的PTEN、p21ras蛋白表达。结果PTEN在正常子宫内膜、不典型增生和内膜腺癌中的阳性表达率逐渐降低,分别为95.0%、40.0%、31.1%。正常子宫内膜组织中的PTEN阳性表达率显著高于不典型增生和内膜腺癌(P<0.01)。p21ras蛋白在正常子宫内膜、不典型增生和子宫内膜腺癌中的阳性表达率分别为0、50.0%、64.4%,子宫内膜不典型增生和子宫内膜腺癌组均显著高于正常子宫内膜组(P<0.01)。PTEN与p21ras蛋白表达呈负相关(r=-0.303,P<0.05),且均与细胞分化程度显著相关(P<0.05)。结论1)PTEN、ki-ras基因在子宫内膜腺癌发生发展中起重要作用,PTEN是其发生的早期分子事件;2)PTEN、p21ras的蛋白表达与病理分级有关;3)在子宫内膜腺癌中PTEN与p21ras可能参与同一蛋白通路发挥生理作用。     

Expression of ras oncogene p21 protein in normal and neoplastic laryngeal tissues: correlation with histopathological features and epidermal growth factor receptors.

Western blotting analysis of the p21 ras oncoprotein was performed in seven normal laryngeal mucosa specimens and 43 primary laryngeal cancers. Varying p21 levels, expressed as optical density (OD), were found in normal mucosa (median 1.94 OD, range 0.90-2.17 OD) and in primary laryngeal tumours (median 1.74 OD, range 0.30-6.37 OD). When p21 expression in laryngeal cancer was compared with the normal counterpart, higher levels were found in neoplastic than in normal laryngeal tissue (median 2.54 OD, range 1.76-6.37 OD, vs median 1.94 OD, range 0.90-2.17 OD) (P = 0.023). Immunohistochemical analysis demonstrated that most of the tumour cells (more than 70%) were immunostained while the stromal component was unreactive. No correlation between p21 expression and tumour location, stage and histopathological grade was observed. The correlation between ras p21 protein expression and epidermal growth factor receptor (EGFR) levels was also investigated. EGFR-positive cases did not show any difference in p21 expression with respect to EGFR-negative cases (median 1.52 OD, range 0.30-6.37 OD, vs median 1.84 OD, range 0.93-3.71 OD). Our findings suggest that overexpression of p21 protein is associated with a malignant phenotype in laryngeal cancer. Further studies should be undertaken to evaluate whether the assessment of p21 protein expression may have clinical significance in laryngeal cancer.     

Quantitation of Harvey ras p21 enhanced expression in human breast and colon carcinomas

Molecular studies have demonstrated increased expression of the Harvey (Ha) ras oncogene in human breast and colon carcinomas. With the use of a direct-binding liquid competition radioimmunoassay (RIA), capable of providing truly quantitative analysis of the 21,000-dalton (p21) ras oncogene and protooncogene products, absolute levels of Ha-ras p21 have been determined in human breast and colon carcinomas, benign lesions, and/or their respective normal tissues. Enhanced Ha-ras expression was documented in 66% of breast and 100% of colon carcinomas as compared with their normal counterparts, with levels in breast carcinomas ranging from 10.1 to 50.4 pg ras p21/micrograms protein and those in colon carcinomas ranging from 18.4 to 51.7 pg ras p21/micrograms protein. Some dysplastic lesions of the breast and colon also contained elevated Ha-ras p21. Relative levels of Ha-ras p21 expression, detected by competition RIA, correlated with percent Ha-ras p21-positive cells as determined by immunohistochemical assays. By use of liquid competition RIA and immunohistochemical assays, it has been shown that levels of ras p21 expression did not always correlate between primary and metastatic colon lesions of the same patient. The use of the quantitative RIA and semiquantitative immunohistochemical assays, in concert with cDNA probes for identification of specific ras point-mutated oncogenes or protooncogenes, may now provide the means for definitive quantitative analyses of ras p21 in human carcinomas and benign lesions.     

Chemopreventive effect of 4'-demethyl epipodophyllotoxin on DMBA/TPA-induced mouse skin carcinogenesis

The chemopreventive effect of topical application of 4'-demethylepipodophyllotoxin (DMEP), an antimitotic agent, on a two-stageskin carcinogenesis model in Swiss Albino mice induced by 9,10-dimethylbenz[a]anthracene(DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated.Two topical applications with 0.24% DMBA over 1 week, followedlater by 5 nmol of TPA twice weekly produced 100% incidenceof tumors in these animals by 18 weeks. Treatment of animalswith DMEP (until the end of the experiment), 30 min before TPAtreatment, significantly reduced the tumor incidence, tumorvolume and the conversion efficiency of papillomas to squamouscell carcinomas. The tumor formation and growth was also delayedby DMEP pre-treatment. Application of DMEP protected againstthe losses provoked in levels of glutathione and activity ofcatalase and superoxide dismutase in skin and liver of animalsby the application of DMBA/TPA. Thus, DMEP might possibly beexerting its chemopreventive activity by acting as an antioxidant. Abbreviations: CAT, catalase; DMBA, 9,10-dimethylbenz[a]anthracene; DMEP, 4'-demethyl epipodophyllotoxin; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; SOD, superoxide dismutase; TPA, 12-O-tetradecanoylphorbol-13-acetate. ² To whom correspondence should be addressed Email: neeta{at}medinst.ernet.in     

Effect of chalones on changes in the rat larynx induced by DMBA

The experiments used 144 non-inbred albino male rats aged 2-3 months. They were divided in 6 study groups and one control group. The animals of groups 1-3 received 0.01 ml infusions of 6.5% solution of DMBA into the larynx twice a week for 6 months. In groups 4-5, animals were treated with acetone and dimethylsulfoxide, respectively. Epidermal chalones were instilled in group 2 1 hr after DMBA infusion, and hepatic ones--in group 3. After 7 and 10 months, the animals were sacrificed with large doses of ether, 6 sections were taken from each larynx and stained with hematoxylin--eosin. The statistically treated results showed chalone treatment to inhibit the development of precancerous lesions in larynx induced by DMBA. The greatest inhibitory effect was registered in epidermal chalone treatment.     

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.     

Ras protein p21 processing enzyme farnesyltransferase in chemical carcinogen—induced murine skin tumors

Farnesylation of ras protein p21 is crucial for the protein's membrane localization, which is essential for its cell-transforming activity, which in turn is thought to be critical for the ultimate induction of cancer. The cytosolic enzyme farnesyltransferase plays a major role in posttranslational modification of p21, but the level of farnesyltransferase activity in mammalian tumors and its relationship to the processing of cytosolic p21 that leads to tumorigenesis are unknown. We report here that farnesyltransferase activity was significantly higher in chemical carcinogen—induced benign skin papillomas in SENCAR mice than in the epidermises of control animals. The enzyme is primarily epidermal in origin, and kinetic studies with cytosol from epidermis and papillomas showed that the reaction was linear with respect to time, substrate concentration, and protein content. Skin papillomas showed significantly elevated levels of both cytosolic and membrane-bound Ha-ras p21, whereas far lesser cytosolic and almost negligible amounts of membrane-bound p21 were present in the epidermis of control mice. There was a positive correlation between increased enzyme activity in papilloma cytosol and the processing of overexpressed cytosolic Ha-ras p21 for its localization to membrane.     

Modulation of DMBA induced genotoxicity in bone marrow by quercetin during skin carcinogenesis

Effect of quercetin was studied on DMBA induced skin carcinogenesis in young adult Swiss albino mice. Quercetin was administered continuously with the diet (2%) for four weeks and chromosomal aberration, a predictor of future cancer risk, studied in the bone marrow cells at different time intervals. Significant reduction of chromosomal aberration was observed in bone marrow after four weeks (P< 0.02). The reduction was first evident after 96 hrs though it was not significant at this stage. Significant decrease occurred from the 21st day onward when quercetin was given in concomitance with 7,12-dimethylbenz[a]anthracene (DMBA) indicating a definite protective effect of quercetin on chromosomal aberration.     

Prognostic significance of ras/p21 alterations in human ovarian cancer.

Ras/p21 oncoprotein expression and K-ras mutations were analysed by Western blot and/or K-ras oligonucleotide hybridization in 78 primary ovarian cancers, 20 omental metastases, two low malignant potential tumours (LMP), nine benign ovarian tumours and 10 normal ovaries. A cut-off value of an integral of absorbance (i.a.) of 2.20, obtained by receiver operating characteristic (ROC) curve, was shown to be the best cut-off for defining p21 positivity. p21 levels were higher in malignant tumours than in benign tumours (median 2.10 i.a. vs median 1.22 i.a.; P = 0.014) and in omental metastases than in primary ovarian carcinomas (median 2.54 i.a. vs median 2.1 i.a.; P = 0.0089). p21 overexpression did not correlate with any of the clinicopathological parameters examined. Follow-up data were available for 63 patients. A significant relationship was shown between p21 positivity and a shorter overall survival (OS) (P < 0.03) and progression-free survival (PFS) (P < 0.03). In multivariate analysis only the presence of ascites, p21 levels and epidermal growth factor receptor status retained an independent prognostic role. K-ras gene mutations were frequently detected in benign and low malignant potential tumours (71.4%), which were mostly mucinous (P = 0.0152).