Ultraviolet B-induced squamous epithelial and melanocytic cell changes in a xenograft model of cancer development in human skin.

We previously demonstrated that precancers (actinic keratoses and dysplasias) and squamous cell carcinomas (SCCs) develop in one quarter of human neonatal foreskins grafted onto recombinase-activating gene-1-knockout mice treated once with 7,12-dimethylbenz[a]anthracene (DMBA) followed by chronic intermediate-range ultraviolet (UV) B light irradiation. The goals of this study were to determine if a longer UVB exposure followed by further observation would increase the number of precancers and invasive cancers and to evaluate whether this model results in changes in p53 expression and cell proliferation similar to those seen in sun-damaged normal skin, actinic keratoses, and SCCs. The treatment consisted of a single dose of DMBA followed by 500 J/m2 UVB radiation administered three times weekly for at least 5 mo. Histologic changes (cysts, hyperplasias, precancers, and/or invasive cancers) were seen in 24 of 25 treated xenografts but not in controls. Ten of 25 grafts (40%) had two or more histological changes, and two human SCCs developed. After seven or more months of UV exposure and a total time from DMBA treatment to killing of 12-18 mo, 83% (15 of 18) of specimens developed squamous precancer or SCC of human origin, and 44% (eight of 18) developed melanocytic hyperplasia or melanoma. The change from moderate dysplasias to SCC required longer UV exposure (median, 11 mo), and 5 mo more observation than did the development of mild dysplasias (median UV exposure, 7 mo; median DMBA to death time, 12 mo). There was a direct correlation between both p53 expression and cell proliferation and the degree of histologic alteration both in squamous epithelial and melanocytic cells.

Analysis of the FHIT gene and its product in squamous cell carcinomas of the head and neck.

The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine if FHIT alterations play a role in human squamous cell carcinogenesis of the head and neck (HNSCC), we examined the gene and its product by RT-PCR, SSCP, Northern, Southern, and Western blot analysis in primary HNSCC and/or HNSCC cell lines. Three of 32 tumor samples lacked detectable expression of FHIT by RT-PCR but showed amplification of a control gene of similar size. One of 29 primary tumors and 2/9 HNSCC cell lines exhibited aberrant transcripts generated by RT-PCR methods using one set of 40 cycles of amplification. FHIT mRNA expression was absent in seven HNSCC cell lines but detectable in primary keratinocytes by Northern analysis. Using specific polyclonal antiserum to the full-length FHIT protein in immunoblot analyses, 4/9 cell lines analysed showed no expression of pFhit, two exhibited low levels of expression, and three expressed a putative truncated pFhit. One of 15 tumors analysed also exhibited an overexpressed truncated protein. PCR/SSCP and Southern analysis of one cell line DNA that expressed a truncated protein indicated that it sustained homozygous loss of FHIT exon 5. Our results suggest that alterations in FHIT at the DNA, RNA, and protein levels exist at a low but significant frequency in HNSCCs. Further studies regarding the potential biological activity of FHIT are needed to clarify the role of this gene in HNSCC tumorigenesis.

Expression of PACE4 in chemically induced carcinomas is associated with spindle cell tumor conversion and increased invasive ability.

Gene expression changes associated with the conversion of squamous cell carcinoma (SCC) to a more advanced malignant spindle cell carcinoma (SPCC) were determined by differential display. Using an animal model of human SCC progression, we provide evidence of increased PACE4 expression in SPCC cell lines and primary tumors induced by chemical carcinogenesis protocols, thus implicating this proprotein convertase in the process of tumor progression. Exogenous overexpression of PACE4 cDNA in mouse SCC cells of low invasive ability resulted in enhanced tumor cell invasiveness that was absent in parental or mock-transfected SCC cells. In addition, the PACE4-transfected cells acquired the ability to process prostromelysin 3 into its active enzyme form. Taken together, these results show that up-regulation of PACE4 expression is associated with SCC conversion to SPCC and suggests that activation of essential PACE4 substrates, such as the metalloproteinase stromelysin 3, is required for tumor cell invasion.

The FHIT gene product is highly expressed in the cytoplasm of renal tubular epithelium and is down-regulated in kidney cancers.

Loss of heterozygosity and homozygous deletion of the 3p14.2 region in human cancers implies the existence of a tumor suppressor gene. One such candidate is the fragile histidine triad (FHIT) gene. To investigate the role of FHIT gene product in tumorigenesis, we generated specific polyclonal antibodies to the human protein and studied its expression in normal and tumor tissues. Immunoblot analysis revealed highly variable expression of pFhit in normal adult human tissues. The highest steady-state level of pFhit was found in kidney and brain, whereas breast, intestine, and skeletal muscle expressed only trace amounts. Within the kidney, the pattern of pFhit immunoreactivity was confined to the tubular epithelium and absent in the glomeruli. Immunofluorescence analysis and biochemical fractionation have sublocalized pFhit to the cytosolic compartment. Compared with normal kidney, pFhit was found to be down-regulated in a subset of primary renal cell carcinoma. Two of 12 renal cell carcinoma cell lines that are known not to contain VHL mutations showed complete loss of pFhit expression. This is supported by the appearance of aberrant reverse transcription-polymerase chain reaction products and loss of the normal-size fragment. Our results are consistent with a potential role of pFhit loss or dysfunction in human renal cell carcinoma independent of VHL involvement.

Similar incidence of K-ras mutations in lung carcinomas of FVB/N mice and FVB/N mice carrying a mutant p53 transgene.

Mutated p53 genes are capable of complementing activated ras genes in the transformation of primary rat embryo fibroblasts in vitro. Mutations in both genes have also been found in several human cancers, including lung carcinomas. We generated transgenic mice containing a p53 construct with a missense mutation in exon 5 (ala135val) to study the role of p53 mutations in lung tumorigenesis, and to facilitate identification of other genetic events that might complement p53 mutations in mouse lung carcinogenesis. The p53 transgenic lines exhibited a higher frequency of lethal lung tumors than the parental FVB/N strain. We examined the spontaneously-arising lung carcinomas from mice expressing the mutated p53 transgene for K-ras mutations using single-stranded conformation polymorphism (SSCP) and/or direct sequencing approaches. Fifteen of 29 (52%) carcinomas contained mutations in the K-ras oncogene. Six of 15 of the K-ras mutations were in codon 61 and 9/15 were in codon 12. Subsequent analysis of spontaneous lung carcinomas from mice of the FVB/N parental strain showed that 9/12 (75%) carcinomas examined contained K-ras mutations. Two of these were in codon 12, one in codon 13, and 6 were in codon 61. These results demonstrate that the frequency of ras mutations does not differ between the p53 FVB/N transgenic mice and their parental FVB/N strain but suggest that a high frequency of mutations K-ras can be correlated with lung tumorigenesis in both groups of mice.

Genetic alterations of p53 and ras genes in 1,3-butadiene- and 2',3'-dideoxycytidine-induced lymphomas.

Mutations of p53 and ras genes were analyzed in 40 and 31 1,3-butadiene (BD)-induced lymphomas of B6C3F1 mice (BLFs), respectively, and in 63 2',3'-dideoxycytidine-induced lymphomas, which were collected from B6C3F1 (n = 16) or NIH Swiss mice (DLSs; n = 47). The frequencies of K- and N-ras mutations in BLFs (32 and 13%, respectively) were higher than those in DLSs (13 and 2%, respectively). Seven of 10 K-ras-mutated BLFs contained codon 13 CGC mutations, whereas no mutation in K-ras codon 13 was detected in DLSs, suggesting that the codon 13 CGC mutation is specific for BD exposure. Interestingly, 8 of 13 BLFs with ras mutations were from low-dose (< or = 200 ppm) or stop-exposure (26 weeks) groups. These results suggest that ras mutations play an important role in the development of BD-induced lymphoma and may represent an early event. Analysis of genetic alterations in exons 5-8 of the p53 gene revealed mutations in seven of the BLFs and three of the DLSs. All seven BLFs carrying p53 mutations were collected from the high-dose (625 ppm) continuous exposure group, which might indicate that p53 is involved in the progression of BD-induced lymphoma and in late stage of lymphomagenesis. Mutations in ras and p53 genes are relatively infrequent in 2',3'-dideoxycytidine-induced lymphomas, suggesting that other genes must be involved.

Increased expression of G1 cyclins and cyclin-dependent kinases during tumor progression of chemically induced mouse skin neoplasms.

Cyclins and cyclin-dependent kinases (Cdks) are central to regulation of the cell cycle. Their abnormal expression may cause loss of cell-cycle control and result in autonomous cell growth, a critical feature of neoplasias. In this study, using immunoblotting, we analyzed the protein levels of several G1/S cyclins (cyclins D1, D2, D3, A, and E) and their respective Cdks (Cdk 2, 4, and 6) in 17 mouse squamous cell carcinomas (SCCs) and 18 mouse skin tumor cell lines. Overexpression of these cell cycle-related genes was frequent in tumors and cell lines. Of special interest was the fact that a group of cell lines that became more aggressive after animal passaging expressed more cyclins D2 and D3 than their respective parental lines did. In addition, SCCs had higher cyclin D3 expression levels than papillomas, and metastases had higher levels than the respective primary tumors, indicating that overexpression of cyclin D3 may be associated with increased aggressiveness of mouse SCC. Interestingly, overexpression of cyclin E was seen in most SCCs induced by a complete carcinogenesis protocol with benzo[a]pyrene (B(a)P) and only in a few SCCs induced by a two-stage carcinogenesis protocol using 7,12-dimethylbenz[a]anthracene as initiator. In contrast, more of the latter tumors overexpressed cyclin D1 and D2 than those induced by B(a)P. Thus, it is possible that different components of the cell-cycle machinery are involved in proliferative dysfunctions that take place during tumor development with different carcinogenesis protocols. Taken together, these results indicate that overexpression of G1 cyclins and their related Cdks is a significant molecular abnormality that could be involved in the process of tumor progression.

The mouse lymphoma L5178Y Tk+/- cell line is heterozygous for a codon 170 mutation in the p53 tumor suppressor gene.

The p53 tumor suppressor protein plays an important role in regulating the cellular response to DNA damage, including cell cycle arrest and apoptosis induction. Normal p53 function is critical for the maintenance of genomic stability. The mouse lymphoma L5178Y/TK(+/-)-3.7.2C cell line is widely used in genetic toxicology for mutagenesis and clastogenesis testing. A related line L5178Y-R, has previously been shown to react with antibodies specific for mutant as well as wild-type p53 protein and to exhibit delayed cell death after radiation. For this reason, as well as the mouse lymphoma assay's reputation for high sensitivity of detection for genotoxic agents but low specificity, we examined several clones of L5178Y cells for mutations in the conserved core domain (exons 5-8) of the p53 gene. Using single-strand conformational polymorphism analysis, we found evidence for the same mutation in exon 5 of p53 in L5178Y-R, L5178Y-S and L5178Y/TK(+/+)-3.7.2C cells. The mutation was identified by sequencing of exon 5 as a TGC (Cys) to CGC (Arg) transition in codon 170 (= codon 176 in humans). Sequencing showed approximately equivalent signals for the mutant and normal alleles for all 3 lines. The mutation in codon 170 is adjacent to a mutation hotspot of the human p53 gene (codon 175) and eliminates a critical zinc-coordinating cysteine residue such that the mutant protein is likely to be denatured and have a dominant negative effect on normal p53 function. Western blots showed approximately 100-fold higher levels of p53 protein in unirradiated L5178Y cells as compared to induced levels of p53 in normal mouse splenocytes 4 h after 5 Gy of gamma radiation. The high levels of p53 protein in L5178Y cells were not further inducible by radiation, whereas an 11-fold induction was seen in the irradiated splenocytes. These results indicate that p53 protein in L5178Y cells is dysfunctional and suggest that this line may therefore be abnormally susceptible to the induction of genetic alterations.

Molecular pathology of primary and metastatic ductal pancreatic lesions: analyses of mutations and expression of the p53, mdm-2, and p21/WAF-1 genes in sporadic and familial lesions.

BACKGROUND: The molecular pathology underlying the development and progression of ductal pancreatic adenocarcinoma is poorly understood relative to that of other major cancers in industrialized societies. The frequency, nature, and distribution of p53 abnormalities, their temporal relationship to the metastatic and clinicopathologic phenotypes of sporadic and familial pancreatic cancer, and their consequent effects on the genetics and expression of critical wild-type p53-regulated genes (mdm-2 and p21/WAF-1) warrant examination in pancreatic adenocarcinoma. This molecular and immunochemical study of the p53, mdm-2, and p21/ WAF-1 genes and gene products examined the largest series of nonneoplastic, neoplastic, and metastatic ductal pancreatic lesions reported to date in relation to clinicopathologic profile. METHODS: Histologically confirmed specimens of primary (n = 136) and metastatic (n = 23) sporadic and familial ductal pancreatic adenocarcinoma lesions were subjected to immunochemical analyses of p53 expression in which a panel of 3 antibodies was utilized. A panel of nonneoplastic but histologically abnormal pancreatic lesions (n = 77) from individuals with varied histories of cigarette smoking were subjected to similar immunohistochemical examinations. In addition, 3 specimens from patients with chronic pancreatitis, 2 specimens of normal fetal pancreata, and 16 specimens of normal adult pancreata were examined as control tissues. Suitable frozen and archival microdissected tumor lesions were evaluated for mutations in exons 4-9 of the p53 gene by single strand conformation polymorphism (SSCP) and dideoxy sequencing analyses in which two distinct sets of outer and nested intron-based amplification primers were used for each exon. A subset of 25 tumor specimens and 18 tumor-derived cell lines for which the p53 mutation status was known were examined for amplification and/or overexpression of the mdm-2 gene; amplification was determined by Southern hybridization and overexpression by immunohistochemical and Western blot analyses. Similarly, mutations in the coding region of p21/WAF-1 gene were examined by SSCP and DNA sequence analyses, and steady-state expression of the p21/WAF-1 protein was assessed by Western blot analysis in these subsets of tumors and tumor-derived cell lines. RESULTS: Positive ductal nuclear p53 immunostaining was demonstrated in 56% of primary tumors and 54% of metastatic lesions. The frequency did not differ significantly between sporadic and familial lesions, and immunostaining was not observed in ductal, acinar, or islet cell elements of normal pancreata or histologically abnormal benign pancreatic lesions from cigarette smokers. A total of 70% of tumor samples revealed reproducible SSCP abnormalities for p53; 42% of these were found in exons 7 and 8. DNA sequence analysis of cases with greater than 35% epithelial cellularity (n = 25) revealed 17 missense mutations, 12 of which were transitions. Seventy-five percent of these transitions were of G:C-->A:T type. A total of 22% of the p53 mutations identified were microdeletions, along with one insertional mutation at exon 8. None of the normal pancreata from sporadic or familial lesions revealed germ-line p53 alterations. Moreover, the frequency and spectra of p53 alterations exhibited no clear, statistically significant association with tumor grade, TNM stage, or patients' cigarette-smoking histories. The mdm-2 gene was neither amplified nor overexpressed immunochemically in a subset of ductal adenocarcinomas, and there was no clear relationship between the p53 mutation status and the status of the mdm-2 gene or protein. Similarly, SSCP and DNA sequence analysis of the p21/WAF-1 gene revealed only 2 genetic abnormalities in a series of 25 primary tumors and 15 tumor-derived cell lines; 1 of the cell lines also revealed the absence of immunoreactive p21/WAF-1 protein...

Deletion and mutation analyses of the P16/MTS-1 tumor suppressor gene in human ductal pancreatic cancer reveals a higher frequency of abnormalities in tumor-derived cell lines than in primary ductal adenocarcinomas.

The putative tumor suppressor gene p16/CDKN2 encodes a specific inhibitor of cyclin D-cyclin-dependent kinase 4 complexes important in cell-cycle regulation and has been found to be deleted or mutated in a variety of human cancers. Thirty microdissected primary human ductal pancreatic carcinomas from patients not subject to radiotherapy or chemotherapy prior to surgical resection of their carcinomas and 18 human pancreatic carcinoma cell lines were analyzed by single-strand conformation polymorphism (SSCP) and DNA sequence analyses and PCR-based deletion analyses for mutations and homozygous deletions of the p16/CDKN2 gene, respectively. Homozygous deletions of the gene were found in five cell lines, and nonpolymorphic SSCP and DNA sequence alterations were found within exon 1 in four cell lines and exon 2 in three lines, for an overall frequency of deletions and mutations of 66%. In contrast, homozygous deletions of p16/CDKN2 were observed in three primary pancreatic carcinomas, and five primary tumors revealed SSCP and/or sequence abnormalities in exon 1 (one case) and exon 2 (four cases), a mutation and deletion frequency of 27%. Immunoblotting analyses confirmed the absence of p16/MTS-1 expression in actively proliferating cell lines with a homozygous deletion of the gene and low-to-moderate levels of p16/MTS-1 expression in cell lines possessing a normal RB-1 gene or protein. These findings suggest that, although p16/CDKN2 may play a role in the pathobiology of pancreatic cancer, inactivation of this putative tumor suppressor gene occurs more frequently in cell lines than in primary ductal pancreatic carcinomas.

One decade of comparative molecular carcinogenesis.

The field of comparative carcinogenesis has expanded greatly during the last decade. During this decade, the advent of molecular biology techniques allowed the isolation and identification of several oncogenes and tumor suppressor genes. Analysis of genetic alterations in these genes enabled the first dissection and comparison of carcinogenesis pathways in humans and rodents at the molecular level. The results showed that most of the oncogenes-/tumor suppressor genes found to be altered in humans were also altered in rodents. Even the molecular pathways involved in carcinogenesis appear to be similar in some organs. Unfortunately, there are still many unknown steps in the process of carcinogenesis. However, overall, the results still indicate that in spite of the differences between rodents and humans, the use and comparison of rodent models with human tumorigenesis is one of the best ways to 1) examine the mechanisms of carcinogenesis, 2) to identify potential carcinogenic compounds, and 3) to help determine potential carcinogenic risk for humans. The potential validity of the comparative carcinogenesis approach should become even more valuable as it becomes more fine-tuned due to the application of new approaches and the identification of new genes for study. The rapid pace of genomic mapping, the use of loss of heterozygosity studies, and the use of genetically susceptible populations (rodents and humans) has and will continue to allow the localization, isolation, and identification of new cancer genes. As each gene is analyzed in human and rodent tumors, the molecular pathway comparisons will become more accurate and detailed. This combined with molecular epidemiological and transgenic approaches will assure that the field of comparative carcinogenesis will continue to grow and be important in the next decade.

Lack of concordant p53 mutations in some paired primary and metastatic mouse squamous cell carcinomas induced by chemical carcinogenesis.

We studied the frequency and pattern of p53 mutations in 16 mouse skin primary squamous carcinomas induced by chemical carcinogens and their 19 matched metastases. The molecular changes were analyzed by polymerase chain reaction-single-strand conformation polymorphism and subsequent direct sequencing analysis. Eleven mutations of the p53 gene were detected in a total of eight primary tumors, and 10 mutations were detected in nine metastases. Only four pairs had identical mutations in primary and paired metastatic tumors. Eight mutations in six pairs were detected in primary tumors but not in their metastases, and four mutations from three matched pairs were detected in metastases but not in primary tumors. The four pairs that contained the same mutations in both the primary and secondary tumors had lymph-node metastases, and all mutations occurred in exon 8. Conversely, five of six pairs with p53 mutations only in primary tumors had lung metastases, and only one of the mutations occurred in exon 8. None of the mutations found only in metastases were located in exon 8. These data indicate that p53 mutations are prevalent in lymph-node metastases and infrequent in lung metastases of mouse skin tumors and that primary tumors with exon 8 mutations may be more likely to metastasize to the lymph nodes.

Activation of H-ras is prevalent in 1,3-butadiene-induced and spontaneously occurring murine Harderian gland tumors.

Treatment of B6C3F1 mice with concentrations of 62.5-625 p.p.m. 1,3-butadiene by inhalation for up to 2 years causes a significantly increased incidence of Harderian gland (HG) neoplasms over untreated controls (Melnick,R., Huff,J., Chou,B.J. and Miller,R.A. Cancer Res., 50, 6592-6599, 1990). Since a specific K-ras mutation (codon 13 GGC-->CGC) had previously been described in lung and liver tumors from 1,3-butadiene-treated B6C3F1 mice, we analyzed 23 adenomas and six adenocarcinomas of the HG from mice exposed to 1,3-butadiene for this mutation and mutations in the H-ras gene. We also examined ras activation in 16 spontaneously occurring HG adenomas and one adenocarcinoma. DNA samples were prepared from paraffin-embedded tissues and analyzed by PCR followed by direct sequencing methods. Only one 1,3-butadiene-induced HG tumor contained the K-ras codon 13 mutation previously detected in lung and liver tumors. However, 16/29 HG tumors from the treated B6C3F1 mice contained H-ras codon 61 mutations. The mutations detected were: 12 CAA-->CGA transitions, two CAA-->CTA and two CAA-->AAA transversions. Eleven of 17 spontaneous HG tumors contained mutations in H-ras codon 61: five CAA-->CGA transitions, two CAA-->CTA transversions and four CAA-->AAA transversions. While the spectrum of ras mutations did not differ between the spontaneously occurring and chemically induced tumors, these data indicate that activation of H-ras contributes to the process of HG tumorigenesis in both groups of these neoplasms.

Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck.

Sixty-eight primary head and neck squamous cell carcinomas and nine head and neck squamous cell carcinoma cell lines were examined for mutations and homozygous deletions of the p16/CDKN2 gene. Homozygous deletions of the p16/CDKN2 gene were found in three lines, and a mutation was detected in another cell line. In contrast, none of the primary tumors showed homozygous deletions and 11 of 68 tumors had missense or nonsense base changes. Seven tumors contained somatic mutations. Five tumors, including one that also had a somatic mutation, had a probable polymorphism at codon 140 leading to an amino acid change from Ala to Thr. Three of these also contained an apparent polymorphism at codon 98, which did not lead to an amino acid change. The frequency of mutations and deletions detected differs markedly between cell lines (44%) and primary tumors (10%) suggesting that while p16/CDKN2 may play a role in tumorigenesis in some head and neck squamous cell carcinomas, inactivation of p16/CDKN2 probably occurs more frequently in cell lines as a result of adaptation to cell culture.

p53 protein accumulation and gene mutation in the progression of human prostate carcinoma.

BACKGROUND: Nuclear accumulation of p53 protein has been shown to be strongly associated with missense p53 mutations. Studies of nuclear accumulation of p53 protein in prostate carcinoma cells have to date been confined to material from primary tumors. PURPOSE: We studied the accumulation of p53 protein in specimens obtained from primary and metastatic sites of prostate carcinoma. By examining the accumulation of this protein as a function of stage, histologic grade, and androgen responsiveness of the tumor, we hoped to determine the role of p53 mutation in the progression of prostate carcinoma. METHODS: The accumulation of the p53 protein in the cell nuclei was determined by immunohistochemical methods using polyclonal antibody to human p53 CM-1. The material studied consisted of formalin-fixed, paraffin-embedded tissue obtained from primary tumors and metastases of 92 patients with prostate carcinoma. Twelve samples from 11 patients were analyzed for the presence of mutations within exons 5-8 of the p53 gene (also known as TP53) by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) analysis. Sequence analysis was subsequently performed on DNA obtained by polymerase chain reaction amplification of PCR-SSCP reactions produced from six different specimens. The chi-square test, Fisher's exact test, and the Freeman Halton test were used for statistical analyses of the results. RESULTS: All tumors with p53 accumulation were metastatic (stage D), poorly differentiated, and androgen independent. Nuclear accumulation of p53 protein was strongly associated with stage (D2 versus D1 versus A-C, P < .0001), grade (Gleason score 8-10 versus 5-7, P < .003), and androgen sensitivity (androgen independent versus dependent, P < .0001). Logistic regression analysis demonstrated that androgen sensitivity predicted p53 outcome better than did stage (P < .0001) or grade alone (P < .006). There was a perfect concordance between the results obtained by PCR-SSCP analysis and the p53 protein accumulation determined by immunohistochemistry in the 12 samples studied. Mutation of the p53 gene was confirmed by sequencing DNA obtained from six specimens positive in the PCR-SSCP assay. CONCLUSIONS: p53 gene mutation is a late event in the progression of prostate cancer and is associated with advanced (metastatic) stage, loss of differentiation, and the transition from androgen-dependent to androgen-independent growth. IMPLICATION: Testing of prostate cancer biopsy specimens from metastatic sites for p53 protein accumulation and gene mutation may provide useful prognostic information and could influence the recommended course of treatment.

Benzo[a]pyrene-induced murine skin tumors exhibit frequent and characteristic G to T mutations in the p53 gene.

Human tobacco-related cancers exhibit a high frequency of G to T transversions in the mutation hot spot region of the p53 tumor suppressor gene, possibly the result of specific mutagens in tobacco smoke, most notably benzo[a]pyrene (B[a]P). No in vivo animal model of B[a]P-induced tumorigenesis has been used, however, to substantiate these molecular epidemiological data experimentally. Direct DNA sequence analysis of the hot spot region (exons 5-8 inclusive) of murine p53 was performed in 20 skin tumors induced by a complete carcinogenesis protocol with B[a]P. Sequence analyses revealed numerous heterozygous missense mutations in carcinomas, specifically in exons 7 and 8 of the p53 gene, and targeting exclusively guanine residues. Moreover, 70% (5/7) of the mutations characterized were G to T transversions. In contrast, direct DNA sequence analysis of 36 skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in either a complete carcinogenesis protocol or in a two-stage carcinogenesis protocol revealed a 30% frequency of heterozygous p53 mutations, with the majority of mutations found in carcinomas, but only a single G to T transversion (1/8). Thus, while mutation frequencies are similar, the pattern and type of p53 mutations in B[a]P-induced skin tumors differs significantly from the mutation spectra in DMBA-induced squamous neoplasias. These in vivo findings in B[a]P-induced tumors lend support to in vitro and molecular epidemiological evidence, suggesting that the p53 tumor suppressor gene may be a selective target of metabolically activated B[a]P species etiologically associated with human tobacco-related cancers.

Molecular and genetic analysis of liver oncogenesis in transforming growth factor alpha transgenic mice.

Overexpression of a transforming growth factor alpha (TGF-alpha) transgene induced the development of liver tumors in 69 of 93 (74%) adult male mice. To identify factors associated with oncogenesis, liver tumors from transgenic animals were characterized at the molecular level. TGF-alpha RNA transcripts were elevated in 17 of 25 (68%) liver tumors, relative to adjacent nontumorous tissue. Expression of the endogenous c-myc and insulin-like growth factor II genes was enhanced in 7 of 19 (37%) and 12 of 16 (75%) tumors, respectively. In contrast, epidermal growth factor receptor RNA levels were unchanged or reduced in all liver tumors, and mutations were not detected in either the Ha-ras or Ki-ras genes. The occurrence of liver tumors in castrated TGF-alpha transgenic mice was reduced about 7-fold, while in ovariectomized transgenic animals the incidence was increased about 6-fold. The progeny of a cross between CD1-derived TGF-alpha transgenic (MT42) and C57BL/6 mice exhibited no reduction in tumor burden (83%); however, the incidence of tumor formation in MT42 x FVB/N offspring was substantially lower (19%). We conclude that in these transgenic mice TGF-alpha promotes tumor formation and appears to play a major role in tumor progression. Moreover, other factors that may collaborate in TGF-alpha-induced hepatocarcinogenesis include c-myc, insulin-like growth factor II, sex hormones, and the genetic background upon which the transgene operates.

Polymerase chain reaction/sequencing analysis of ras mutations in paraffin-embedded tissues as compared with 3T3 transfection and polymerase chain reaction/sequencing of frozen tumor deoxyribonucleic acids.

The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.

Murine p53 intron sequences 5-8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumors.

Inactivating point mutations and small deletions in the p53 tumor suppressor gene have been found in human liver and lung tumor--derived cell lines and tumors. However, little evidence has been reported concerning inactivation or mutation of the p53 gene in mouse primary tumors. To examine CD-1 mouse liver and lung tumors for mutations in the p53 gene, we first sequenced p53 introns 5-8 so that polymerase chain reaction amplification and sequencing primers located within the introns could be prepared. Use of these primers prevented amplification of the mouse p53 pseudogene and allowed sequencing of exons 5-8 in their entirety as well as their intron-exon junctions. DNA isolated from CD-1 mouse tumors was amplified and directly sequenced using nested primers. Nine spontaneous hepatocellular carcinomas (HCCs) and 34 chemically induced HCCs (induced by single intraperitoneal injections of N-nitrosodiethylamine [DEN] [8 HCCs], 7,12-dimethylbenz[a]anthracene [DMBA] [8 HCCs], 4-aminoazobenzene [8 HCCs], and N-OH-2-acetylaminofluorene [10 HCCs]) were examined for mutations in exons 5-8 of the p53 gene. In addition, 12 spontaneous, 10 DMBA-induced, and 13 DEN-induced lung adenocarcinomas or adenomas were analyzed for mutations. No mutations were found in any of the tumors examined. However, a mutation was demonstrated at codon 135 in the positive-control plasmid LTRp53cG(val). The results of this study suggest that inactivation of p53 is unlikely to play a major role in murine lung or liver carcinogenesis. However, inactivation of p53 may occur at a very low frequency, or it may occur as a late event and therefore be present in only a very small number of the tumor cells, rendering it undetectable by this method. Lastly, although few p53-inactivating mutations are found outside of exons 5-8 in human tumors, it is possible that these murine tumors contained mutations outside of this region and were therefore missed by our approach.