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Tumor suppression by p53 is deficient in the majority of human cancers. Previous studies have suggested that expression of p53 in human cancer cells can result in either growth arrest or apoptosis. The biological and genetic de-terminants that dictate which of these two pathway - apoptosis or arrest - will be chosen by a particular cell following p53 expression are largely un-known. To investigate the basis of this difference, we evaluated the role of p21, a mediator of p53-induced growth arrest. We generated a replication-deficient adenoviral recombinant which expresses p21 and com-pared its tumor suppressive abilities with Ad-p53. Infection with Ad-p21 re-sulted in high levels of p21 expression and suppressed the growth of human cancer cells, through the Gl arrest of the cell cycle. We then examined the effects of combined infection with Ad-p21 and Ad-p53 to investigate which of these molecules had the dominant function. Introduction of exogenous p53 in RERF-LC-OK, BT549 and ZR-75-1 cells overcame p21-mediated cell cycle arrest at G1 and induced apoptosis, suggesting that this affect is a general event among human cancer cell lines. We then evaluated the role of Bax/Bcl-2 in the response to p53. A Significantly greater amount of Bax protein was pre-sent in cell lines undergoing apoptosis than in cells with arrested growth,suggesting that Bax might be an important component of the p53-mediated apoptosis of cancer cells.

Primary baby rat kidney (BRK) cells were transfected with two different non-immortalizing mutants of adenovirus type 5 (Ad5) E1A(s) and/or human papillomavirus type 16 (HPV16) E7(s) to test whether immortalization was induced by the genetic complementation. Co-transfection of an RB-nonbinding E1A mutant (d1922/947) and N-terminal E7 mutant (2PRO) formed transformed foci on BRK cells, but a combination of an N-terminal E1A mutant (NTdl598) and RB-nonbinding E7 mutant (24GLY) did not. However, NTd1598 plus 24GLY as well as dl922/947 plus 2PRO produced immortalized BRK cell lines. The cell lines immortalized by dl922/947 plus 2PRO showed a morphology similar to those immortalized by Ad5 E1A ; while cell lines immortalized by NTdl598 plus 24GLY showed a morphology similar to those immortalized by HPV16 E7. These results suggest that the RB-binding regions of Ad5 E1A and HPV16 E7 are mutually replaceable, and that the N-terminal function of E1A is essential for focus formation and maintenance of transformation morphology. A combination of RB-nonbinding mutants, E1A dl922/947 plus E7 24GLY, was able to immortalize BRK cells when introduced together with the Ad5 E1B 19K gene. The Ad5 E1B 55K and Bcl-2, which also possess anti-apoptotic activity, did not increase colony formation of and/or immortalize BRK cells when co-transfected with dl922/947 plus 24GLY. In a CAT assay E1B 19K, but not Bc1-2, enhanced E2F-dependent CAT transactivation by Ad5 ElA or dl922/947. Thus, it is suggested that E1B 19K might complement RB-nonbinding mutants by means of two activities, suppression of apoptosis and enhancement of E2F-dependent transactivation.

p53 gene mutation and its protein accumulation are widely detected in human cancers and have prognostic significance. To examine the relationship between p53 mutation and its protein accumulation, tumor samples from 65 oral cancer patients were analyzed using both single strand conformational polymorphism (SSCP) technique to screen the presence of p53 gene mutations and immunohisto-chemistry to detect the p53 protein accumulations. Results were simultaneously associated with clinicopathological variables of the cancers and prognoses of the patients. p53 gene mutations were found in 18 cancers (7.6%), whereas aberrant accumulations of p53 protein were present in 31 cancers (47.6%). All of the oral cancers with p53 mutation showed positive immunoreactivity, but in addition, p53 protein accumulations were still found in 13 oral cancers (20%) without detect-able gene mutations. The concordance between the protein expression and the SSCP analysis was 80%. Such p53 abnormalities were significantly correlated with lymph node metastasis and clinical stage. Patients with the detectable p53 abnormalities survived for a significantly shorter period of time. p53 abnormal-ities are reliable as an indicator for evaluating malignant potential of oral can-cers.

HPV infection has long been implicated in the development of cervical car-cinoma. We have analyzed the HPV 16 genome structure and expression of the viral mRNA in cervical intraepithelial neoplasias (CINs) and cervical car-cinomas by using modified polymerase chain reaction (PCR) methods. Genome structure has been determined by PCR using multi-primer sets which are located in each open reading frames and then these results have been compared with the physical state of the viral DNA determined by two-dimensional electrophoresis. Furthermore, we have analyzed the expression of HPV 16 mRNA and genome structure using DNA and RNA simultaneously extracted from CINs and cervical carcinomas using PCR and reverse transcription (RT-) PCR. Our data showed that the DNA regions from the El to Ll region were delet-ed in two of three CINs containing episomal HPV 16 and three out of seven cervical carcinomas containing integrated HPV 16. However, the E6/E7 region was conserved in all the HPV 16-positive samples. RT-PCR analysis has determined the presence of mRNA species which could encode the E6, E6*I, E6*II, E7, E2, E2 ? C, E1^E4, E1^E2 ? C, E4, E2 ? C-E5 and L2 proteins. The overall results of DNA and mRNA analyses in cervical lesions indicated that the expression patterns of the early and late transcripts studied were not specifically related to the grade of malignancy and the physical state or the deletion of the viral genome. Furthermore, alterations in the splicing pat-terns of HPV 16 transcripts may not be involved in tumor progression.

Specific types of human papillomaviruses (HPVs) are closely associated with the development of genital carcinomas. We previousy reported a PCR method which amplifies the E6E7 sequence from 6 different high-risk genital HPVs (HPV16, 18, 31, 33, 52 and 58) (J Gen Virol 1991, 72 : 1039-1044.). To amplify broader types of genital high-risk HPVs, we have modified our consensus primers by extending 9 nucleotides at the 3 end of the sense primer and changing 5 nucleotides at the 5 end of the anitisense primer. Genotype diag-nosis was carried out by AvaII plus Rsal digestion. This modified PCR method enabled the detection of trace levels of at least 11 types of genital high-risk HPVs (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56 and 58), at subpicogram to sub-nanogram amounts of cloned DNA, amplified after the consensus PCR. We applied this method to analyze 155 cervical scrapes from patients who had been diagnosed with premalignant or malignant cervical lesions. HPVs were detected in 63.0% of mild dysplasia (17/27), 100% of moderate dysplasia (all 12 cases), 91.7% of severe dysplasia (11/12), 95.8% of carcinoma in situ (23/24), and 80.0% of invasive cervical cancer (52/65). HPV16 was present predominant-ly (60.9%), followed by HPV58 (153%), HPV52 (13.9%), HPV31 (13.0%), HPV18 (6.1%), HPV35 (2.6%), HPV51 (2.6%), HPV56 (2.6%), HPV33 (1.7%) and HPV39 (1.7%). Five cases contained unclassified types (4.3%). The results indicate that this modified E6E7 consensus PCR method provides a quick and easy way to detect and diagnose genotypes of the high-risk genital HPVs from scraped cells.

Human papillomavirus type 16 (HPV16) is involved in the development of cervical carcinoma and its viral gene products E6 and E7 are believed to be essential for the carcinogenic process. We analyzed the effect of retinoic acid (RA) on the growth and/or HPV16 E6 and E7 gene expression in the HPV16-containing cervical carcinoma cell lines SiHa, CaSki and QG-U and the HPV-free cell lines C33A and EBC-1. RA (10-6 M) suppressed the growth of SiHa cells by more than 90% and of QG-U cells by about 40% ; however, the growth of CaSki, C33A and EBC-1 cells was not affected by RA. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was applied for investigation of the relationship between RA and the level of HPV16 E6/E7 mRNA. As a result, the E6/E7 mRNA level in SiHa and QG-U was reduced by RA by about 60% and 75%, respectively, while RA had no obvious effect on the E6 and E7 gene expression in CaSki cells. The chloramphenicol acetyltransfer-ase (CAT) assay showed that transcription driven by HPV16 non-coding region (NCR) was suppressed by RA by about 75%. These results suggest that at least a part of the growth suppression of SiHa and QG-U by RA is mediated by sup-pression of E6 and E7 expression.

Expression of c-ras oncogene was analyzed in 75 cases of carcinomas, 38 benign lesions and 22 normal mucosae of the stomach in Chinese subjects by use of the monoclonal antibody rp28, which reacts to the c-Ha-ras and c-Ki-ras p21 products. The incidence of p21 positive cases (more than 20% of total cells stained by rp28) was 90.7% in gastric carcinomas, 10.5% in benign gastric lesions and 4.5% in normal mucosae. While 34/75 gastric carcinomas contained strongly rp28-reactive (+ +) cells, none of the benign lesions or normal mucosae did. The mean percentage of rp28 reactive (+ or ++) cells in each group was 52.2% in gastric carcinomas, 6.3% in benign lesions and 2.3% in normal mucosae. These indicate that ras p21 expression is significantly high in the gas-tric carcinomas compared to the benign lesions and normal mucosae of the stom-ach. Tests of tubular adenocarcinomas with different degrees of differentiation showed significantly higher amounts of p21 product in well differentiated (71.7%) and moderately differentiated (67.3%) samples than in poorly differentiated adenocarcinomas (40.9%), which contained more, in tern, than mucinous adenocarcinomas (39.2%) and undifferentiated carcinomas (27.5%). This sug-gests that Ha- and/or Ki-ras p21 expression may have some roles in the mainte-nance of the glandular structure in gastric carcinomas.

We studied the effects of murine wild-type and mutant p53 genes (p53-wt and p53val135) on the growth and transformation of murine osteoblastoid cell line MC3T3-El. The mutant p53val135 enhanced focus formation of MC3T3-E1 cells by the activated H-ras plus LTR-myc gene and H-ras plus adenovirus 12 E1A gene more than four fold each, while p53-wt suppressed them 0.4 and 0.3 fold, respectively. The plating efficiency of hygromycin-resistant MC3T3-E1 cells after transfection of pSV2hygro were also increased by more than three fold with the cotransfection of p53val135 and the efficiency was also decreased 0.2 fold by cotransfection of p53-wt. These indicate that p53val135 enhances and p53-wt suppresses not only oncogene focus formation but also the cellular growth of the murine MC3T3-E1 cell line. Southern blot hybridization detected the tran-sfected p53-wt sequence only in three out of ten MC3T3-E1 cell lines established from foci induced by p53-wt and oncogenes, and failed to detect the p53-wt DNA in hygromycin-resistant MC3T3-E1 cell lines transfected with pSV2hygro and p53-wt. These suggest that MC3T3-E1 cells containing p53-wt are at a dis-advantage to form transformed foci or colonies, and suggests that MC3T3-E1 provides a good in vitro system to test the biological activity of murine wild-type and mutant p53 genes.

Twenty-six lung cancer tissues from operation or autopsy and eight lung cancer cell lines were examined for an amplification of the ten different cellular oncogenes, c-myc, N-myc, L-myc, c-erbB-1, c-erbB-2, c-fos, v-sis, c-H-ras, v-K-ras, and N-ras, by the Southern blot hybridization. In small cell lung cancer(SCLC), one SCLC tissue(13-1) out of four SCLC tissues and one SCLC cell line contained three fold amplifications of the L-myc oncogene. In non-SCLC, one adenocarcinoma cell line(SLO51) and one large cell carcinoma cell line(SLC-30) showed an amplification of the c-myc oncogene at four fold normal amount and twenty fold amplifications of the c-erbB-1 oncogene respectively, out of twenty-two non-SCLC tissues and seven non-SCLC cell lines. Other lung cancer tissues and cell lines did not show any detectable amplifications or rearrangements of oncogenes. These results suggest that an amplification of the studied oncogenes may not always be associated with carcinogenesis even in lung cancer cell lines. Compared with lung cancer cell lines, the frequency of an amplification of oncogenes in lung cancer tissues was low, especially in non-SCLC tissues.