The frequency of p53, K-ras mutations, and microsatellite instability differs in uterine endometrioid and serous carcinoma: evidence of distinct molecular genetic pathways.

BACKGROUND: The two most common types of uterine endometrial carcinoma, endometrioid (UEC) and serous (USC), differ in their histopathologic appearance and biologic behavior. Recent studies suggest that these differences may be associated with distinct molecular genetic alterations. METHODS: In the current study, the authors compared the frequencies of K-ras and p53 mutations and microsatellite instability (MI) between UEC and USC by analyzing all 3 molecular genetic changes in one set of tumors. Furthermore, the distribution of these molecular genetic alterations was determined among UECs of different histopathologic grade. The authors analyzed 58 UECs with known MI status for K-ras and p53 mutations. The K-ras and p53 genes were analyzed in 45 and 6 cases of USC, respectively. These results were combined with previous data on p53 mutations (21 cases) and MI (34 cases) in USC. RESULTS: MI was present in 16 of 57 UECs (28%) but in none of 34 USCs. p53 mutations were found in 7 of 42 UECs (17%) and 25 of 27 USCs (93%) by direct sequencing of exons 5-8. UECs and USCs with p53 mutations showed strong immunoreactivity for p53 in about 85% of the cases, whereas about 15% of the cases were immunonegative. K-ras mutations at codon 12 were found in 15 of 58 UECs (26%) and in only 1 of 45 USC (2%) by dot blot oligohybridization after polymerase chain reaction amplification of exon 1. Notably, the frequency of both K-ras and p53 mutations and MI was significantly different between UEC and USC (P < 0.001). In UECs, MI and K-ras mutations occurred in low grade as well as in high grade tumors, whereas p53 mutations were present almost exclusively in high grade tumors. CONCLUSIONS: The results of this study suggest that different molecular genetic pathways are involved in the pathogenesis of UEC and USC and that low grade UEC may progress to high grade UEC. These findings support the hypothesis that UEC and USC are separate entities and suggest that different molecular genetic alterations may be responsible for their distinct morphology and biologic behavior.

Reproducibility of the diagnosis of endometrial hyperplasia, atypical hyperplasia, and well-differentiated carcinoma.

Many studies have attempted to identify histologic features that aid in the distinction of atypical hyperplasia (AH) from hyperplasia without atypia and well-differentiated endometrioid carcinoma, but few have evaluated the reproducibility of these diagnoses. Five pathologists independently reviewed 100 endometrial biopsy and curettage specimens chosen to represent the entire spectrum of proliferative lesions of the endometrium, including proliferative endometrium (PEM), hyperplasia without atypia, AH, and well-differentiated endometrioid carcinoma. Slides were reviewed twice for diagnosis, with an intervening evaluation of a checklist of histologic features. Intraobserver and interobserver agreement were assessed using the kappa statistic. Intraobserver kappa values ranged from 0.67 to 0.89 (76% to 89% agreement). Interobserver kappa values by diagnostic category were: proliferative endometrium: 0.86; hyperplasia without atypia: 0.60; AH: 0.47; well-differentiated endometrioid carcinoma: 0.83; with a kappa value of 0.69 for all cases combined. Associations between the selected histologic features and the given diagnoses for each pathologist were analyzed using multiple logistic regressions to identify features that were useful for distinguishing among diagnostic categories. Histologic features determined by univariable and multivariable analyses that were found to be most associated with distinguishing diagnostic categories were: proliferative endometrium versus hyperplasia without atypia: gland crowding (univariable, multivariable), and gland branching (univariable); hyperplasia without atypia versus AH: presence of nucleoli (univariable, multivariable), nuclear enlargement (univariable), vesicular chromatin change (univariable), nuclear pleomorphism (univariable), chromatin irregularities (univariable), and loss of polarity (univariable); hyperplasia without atypia versus carcinoma: glandular confluence/complex cribriform pattern (univariable, multivariable), stromal alteration (univariable, multivariable), and necrosis (univariable). In summary, interobserver agreement was good but was lowest for AH. Only the presence of nucleoli was strongly associated with distinction of AH from hyperplasia without atypia. Individual pathologists use additional features to diagnose atypia, but these features are not consistently associated with that diagnosis. Cribriform architectural pattern and stromal alteration were associated with the distinction of well-differentiated endometrioid carcinoma from AH.

DCC genetic alterations and expression in endometrial carcinoma.

DCC (Deleted in Colorectal Carcinoma) is a candidate tumor suppressor gene located on the long arm of chromosome 18. DCC was initially identified and cloned during a search for the target gene located in a region of 18q that demonstrated loss of heterozygosity (LOH) in 70 to 80% of colorectal cancers. More recently, the region of 18q harboring the DCC gene has been shown to undergo LOH in approximately 14 to 30% of endometrial carcinomas. These findings suggest that DCC may be a target of LOH in at least some endometrial carcinomas and, therefore, may have a role in the pathogenesis of this common malignancy of the female genital tract. To address this possibility, we analyzed 26 cases of endometrioid endometrial carcinoma for DCC LOH and alterations in an AT microsatellite repeat located in an intron of the DCC gene. LOH was detected in one case (4%). Allelic shifts at the DCC AT repeat were detected in five (19%) additional cases. We also evaluated DCC protein expression by immunohistochemical analysis in normal, hyperplastic, and neoplastic endometrial tissues. Three proliferative and five secretory endometria and one simple endometrial hyperplasia demonstrated staining for DCC. Four of the 26 endometrioid endometrial carcinomas for which frozen tissue was available, including at least one from each histologic grade, and a case of endometrioid carcinoma confined to the endometrium completely lacked detectable staining for DCC. Although DCC LOH was infrequent in endometrial carcinomas, alterations of the gene (LOH or AT repeat alterations) were not uncommon (23% of our cases). In addition, DCC was expressed in normal endometrial tissue, whereas expression was lost in all of the five endometrial carcinomas. The combination of the genetic alterations and loss of DCC protein expression suggests that inactivation of the DCC gene may play a role in the pathogenesis of endometrial carcinoma.

Microsatellite instability is uncommon in uterine serous carcinoma.

Thirty-four uterine serous carcinomas, a type of endometrial carcinoma with aggressive behavior and a high frequency (90%) of p53 gene mutations, were analyzed for microsatellite instability (MI). Genomic DNA isolated from paired normal and tumor tissue was analyzed at eight microsatellite loci (D2S119, D2S123, D2S147, D10S197, D13S175, D18S58, D18S69, and ATn) located on four different chromosomes. All 34 tumors failed to meet the criteria for MI, defined as an alteration in the size of at least two of the microsatellite loci in tumor DNA when compared with normal DNA. Only three tumors demonstrated a shift in the size of a single microsatellite locus. Previously we reported MI in 20% of uterine endometrioid carcinomas, the most common type of endometrial carcinoma. The observed difference in the MI frequency between endometrioid and serous carcinoma is statistically significant (P = 0.003). Our data demonstrate that MI is uncommon in uterine serous carcinoma and support that different pathogenetic mechanisms are involved in the development of the two most common types of endometrial carcinoma.

p53 gene mutations are common in uterine serous carcinoma and occur early in their pathogenesis.

Uterine serous carcinoma (USC) is an uncommon but aggressive type of endometrial cancer associated with rapid progression of disease and a poor prognosis. Both USC and its recently described putative precursor, endometrial intraepithelial carcinoma (EIC), demonstrate strong p53 overexpression by immunohistochemistry, suggesting alteration of the p53 gene in their pathogenesis. In the present study, we evaluated 21 USCs and 9 EICs for mutations in the p53 gene using direct sequence analysis and found that 90% of USCs and 78% of EICs contain mutations. Significantly, mutations were found in 3 cases of EIC without associated invasive carcinoma and identical mutations were detected in cases with synchronous USC and EIC. Strong p53 immunoreactivity was seen in the majority of USCs and EICs and correlated with p53 gene mutation, although lack of reactivity did not always indicate the absence of a gene mutation. Loss of heterozygosity of chromosome 17p was observed in 100% of USCs and in 43% of EICs, demonstrating that loss of the wild-type p53 allele occurs early in the development of serous carcinoma. Overall, our results reveal that p53 mutations are very common in USC and EIC. The presence of p53 gene mutations in EIC further suggests that p53 alteration plays an important role early in the pathogenesis of serous carcinoma, possibly accounting for its aggressive biological behavior.

Human papillomavirus E6 and E7 oncoproteins alter cell cycle progression but not radiosensitivity of carcinoma cells treated with low-dose-rate radiation.

PURPOSE: Low-dose-rate radiation therapy has been widely used in the treatment of urogenital malignancies. When continuously exposed to low-dose-rate ionizing radiation, target cancer cells typically exhibit abnormalities in replicative cell-cycle progression. Cancer cells that arrest in the G2 phase of the cell cycle when irradiated may become exquisitely sensitive to killing by further low-dose-rate radiation treatment. Oncogenic human papillomaviruses (HPVs), which play a major role in the pathogenesis of uterine cervix cancers and other urogenital cancers, encode E6 and E7 transforming proteins known to abrogate a p53-dependent G1 cell-cycle checkpoint activated by conventional acute-dose radiation exposure. This study examined whether expression of HPV E6 and E7 oncoproteins by cancer cells alters the cell-cycle redistribution patterns accompanying low-dose-rate radiation treatment, and whether such alterations in cell-cycle redistribution affect cancer cell killing. METHODS AND MATERIALS: RKO carcinoma cells, which contain wild-type P53 alleles, and RKO cell sublines genetically engineered to express HPV E6 and E7 oncoproteins, were treated with low-dose-rate (0.25-Gy/h) radiation and then assessed for p53 and p21WAF1/CIP1 polypeptide induction by immunoblot analysis, for cell-cycle redistribution by flow cytometry, and for cytotoxicity by clonogenic survival assay. RESULTS: Low-dose-rate radiation of RKO carcinoma cells triggered p53 polypeptide elevations, p21WAF1/CIP1 induction, and arrest in the G1 and G2 phases of the cell cycle. In contrast, RKO cells expressing E6 and E7 transforming proteins from high-risk oncogenic HPVs (HPV 16) arrested in G2, but failed to arrest in G1, when treated with low-dose-rate ionizing radiation. Abrogation of the G1 cell-cycle checkpoint activated by low-dose-rate radiation exposure appeared to be a characteristic feature of transforming proteins from high-risk oncogenic HPVs: RKO cells expressing E6 from a low-risk nononcogenic HPV (HPV 11) exposed to low-dose-rate radiation arrested in both G1 and G2. Surprisingly, despite differences in cell-cycle redistribution accompanying low-dose-rate radiation treatment associated with high-risk HPV transforming protein expression, no consistent differences in clonogenic survival following low-dose-rate radiation treatment were found for RKO cell sublines expressing high-risk HPV oncoproteins and arresting only in G2 during low-dose-rate radiation exposure vs. RKO cell sublines exhibiting both G1 and G2 cell-cycle arrest when irradiated. CONCLUSION: The results of this study demonstrate that neither HPV oncoprotein expression nor loss of the radiation-activated G1 cell-cycle checkpoint alter the sensitivity of RKO carcinoma cell lines to low-dose-rate radiation exposure in vitro. Perhaps for urogenital malignancies associated with oncogenic HPVs in vivo, HPV oncoprotein-mediated abrogation of the G1 cell-cycle checkpoint may not limit the potential efficacy of low-dose-rate radiation therapy.

Expression of HPV16 E6 or E7 increases integration of foreign DNA.

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.

Loss of DCC expression in neuroblastoma is associated with disease dissemination.

DCC, a candidate tumor suppressor gene from chromosome 18q21, is most highly expressed in the developing nervous system. In vitro studies suggest a role for DCC in neuronal differentiation, and 18q allelic loss occurs in a subset of neuroblastomas. To address the hypothesis that loss of DCC function may contribute to tumorigenesis in cells of neural origin, we utilized a combination of RNase protection, immunoblotting, and immunohistochemical approaches to characterize DCC expression in 62 primary neuroblastomas and 16 neuroblastoma cell lines. The DCC protein was undetectable in 38% of the primary tumors and 56% of the cell lines. Of note, primary tumors lacking DCC expression were more likely to have been obtained from patients with disseminated or stage D disease (P = 0.01). In addition, loss of DCC expression was observed in three of six primary tumors from stage DS patients. No consistent relationship between the loss of DCC expression and N-myc amplification was observed in our studies. Our findings suggest that loss of DCC expression may contribute to the dissemination of neuroblastoma cells, perhaps through alterations in growth and differentiation pathways distinct from those regulated by N-myc.

DPC4 gene in various tumor types.

We recently identified a novel tumor-suppressor gene, DPC4, at chromosome 18q21.1 and found that both alleles of DPC4 were inactivated in nearly one-half of the pancreatic carcinomas. Here, we analyzed 338 tumors, originating from 12 distinct anatomic sites, for alterations in the DPC4 gene. Sixty-four specimens were selected for the presence of the allelic loss of 18q and were further analyzed for DPC4 sequence alterations. An alteration of the DPC4 gene sequence was identified in one of eight breast carcinomas and one of eight ovarian carcinomas. These results indicate that whereas DPC4 inactivation is prevalent in pancreatic carcinoma (48%), it is distinctly uncommon (< 10%) in the other tumor types examined. The tissue restriction of alterations in DPC4, as in many other tumor-suppressor genes, emphasizes the complexity of rate-limiting checkpoints in human tumorigenesis.

Chromosome abnormalities in primary endometrioid ovarian carcinoma.

Specific and recurrent chromosome abnormalities may occur in regions of the genome that are involved in the conversion of normal cells to those with tumorigenic potential. Ovarian cancer is the primary cause of death among patients with gynecologic malignancies. We performed cytogenetic analysis in a subgroup of epithelial ovarian tumors, the endometrioid tumors, which are histologically indistinguishable from endometrial carcinoma of the uterus. We studied 10 endometrioid tumors to determine the degree of cytogenetic similarity between these two carcinomas. Six of 10 endometrioid tumors showed a near-triploid modal number, and one had a tetraploid modal number. Eight of the 10 contained structural chromosome abnormalities, of which the most frequent were 1p-- (5 tumors), 6q-- (4 tumors), 19q+ (4 tumors), and chromosome 3 rearrangements (4 tumors). These cytogenetic results resemble those reported for papillary ovarian tumors and differ from those of endometrial carcinoma of the uterus. We conclude that despite the histologic similarities between the endometrioid and endometrial carcinomas, the genetic abnormalities in the genesis of these tumors differ significantly.

p53 mutations and clonality in vulvar carcinomas and squamous hyperplasias: evidence suggesting that squamous hyperplasias do not serve as direct precursors of human papillomavirus-negative vulvar carcinomas.

Previous studies of vulvar carcinomas have shown two distinct subsets with respect to several clinicopathologic features. In younger women, the tumors are frequently human papillomavirus (HPV) positive, are usually of basaloid or warty histology, and are associated with vulvar intraepithelial neoplasia. In older women, the tumors are usually HPV negative, are typical keratinizing squamous carcinomas, and are associated with squamous hyperplasia--a lesion that has been purported to serve as a precursor to HPV-negative invasive carcinoma. In squamous carcinomas of the cervix, p53 inactivation (through gene mutation or interaction with the HPV E6 oncoprotein) occurs in most cases. Comparatively few studies have assessed p53 mutation and HPV status in vulvar carcinomas, and none has used molecular markers to evaluate squamous hyperplasias as direct precursors of HPV-negative invasive cancers. Of 18 invasive squamous carcinomas analyzed, seven (39%) were found to be HPV positive. Four p53 gene mutations were identified--all in HPV-negative tumors. DNA was subsequently prepared from microdissected archival tissues from all four specimens showing p53 gene mutations. DNA was separately isolated from normal squamous epithelium, invasive squamous carcinoma, and associated squamous hyperplasia. In each specimen, the p53 mutation was confirmed in the invasive tumor and absent in both normal and hyperplastic epithelium. To further investigate squamous hyperplasia as a potential precursor of HPV-negative invasive carcinoma, the authors determined the clonality of hyperplastic lesions adjacent to invasive carcinomas with p53 mutation. Clonality analyses were performed using a polymerase chain reaction (PCR)-based assay for X chromosome inactivation. Although all three informative carcinomas tested were monoclonal, corresponding normal epithelia and hyperplastic lesions were polyclonal. These findings underscore the heterogeneity of vulvar cancers with respect to loss of wild type p53 function either by interaction with the HPV E6 oncoprotein or somatic mutation of p53, and suggest that squamous hyperplasias do not serve as direct precursors of HPV-negative squamous carcinomas.

Rapid identification of patient specimens with microsatellite DNA markers.

Despite the use of standardized clerical and processing procedures in surgical pathology, questions might arise regarding the proper identification of specimens with respect to patient source. Genotypic analysis of microsatellite DNA polymorphisms was used to identify the patient source of two surgical pathology specimens showing carcinoma. Four highly polymorphic microsatellite loci were evaluated in DNA extracted from various formalin-fixed, paraffin-embedded tissues. Using this technique, we determined that the diagnosis of poorly differentiated adenocarcinoma arising from a background of colitis had been assigned to the correct patient, despite the fact that multiple repeat endoscopic examinations, with biopsy specimens, were negative. In the second case, a suspected processing error involving the exchange of specimen accession numbers was resolved when a lymph node containing a microscopic focus of metastatic carcinoma was assigned to the appropriate patient. A multitude (approximately 50,000 to 100,000) of microsatellite loci are distributed throughout the human genome, and many are highly polymorphic. Hence, genotypic analysis using microsatellite loci has a significantly higher power of discrimination than other commonly used methods. The technique is rapid and is particularly well suited to the analysis of small, fixed-tissue specimens.

Both cell proliferation and apoptosis increase with lesion grade in cervical neoplasia but do not correlate with human papillomavirus type.

Recent molecular studies suggest that the expression of high-risk but not low-risk human papillomavirus (HPV) oncoproteins E6 and E7 can significantly alter normal cell cycle regulation. The alterations in cell cycle regulation may be reflected by changes in the balance between cell growth and cell loss through apoptosis in cell populations expressing E6 and/or E7. We evaluated the kinetic indices of cell proliferation and apoptosis in a histopathological spectrum of cervical neoplasia and compared low-versus high-risk HPV-associated lesions. The cell proliferation index, as determined by detection of the nuclear antigen Ki67, increased with increasing lesion grade. Apoptotic cells were identified with terminal deoxynucleotidyl transferase-labeling of the 3'-hydroxyl ends of DNA nucleosomes. No apoptosis was observed in normal epithelium, and only occasional apoptotic cells were seen in low-grade lesions. However, there was a low but measurable apoptotic index in the higher grade lesions, which increased with lesion grade. There was no significant difference in the proliferative and apoptotic indices in similar grade lesions when stratified into low-versus high-risk HPV types. These findings suggest that apoptosis in HPV-infected lesions correlates with proliferative activity rather than HPV type.

Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency.

A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability.

We have recently demonstrated that mutation of the transforming growth factor-beta (TGF-beta) receptor type II (RII) gene is characteristic of colon cancers exhibiting microsatellite instability or replication errors (RER+). Moreover, we have shown that RII mutations in these RER+ colon cancers are characteristically frameshift mutations within a 10-bp polyadenine repeat present in the RII-coding region. We now show that RII gene mutations in this polyadenine repeat are also commonly present in RER+ gastric cancers (71%). In contrast, we find these same RII gene mutations are distinctly uncommon in RER+ endometrial cancers (17%, P < 0.02). These results suggest that RII gene mutations confer a growth advantage and are selected for in RER+ cancers of both the upper and lower gastrointestinal tract. The genesis of RER+ endometrial tumors must, however, be by a different route.

Mutations in DNA mismatch repair genes are not responsible for microsatellite instability in most sporadic endometrial carcinomas.

Endometrial carcinoma is the second most common tumor type in women with hereditary nonpolyposis colorectal carcinoma. Microsatellite instability (MI) has been observed in the inherited (hereditary nonpolyposis colorectal carcinoma-associated) form of endometrial carcinoma as well as in approximately 20% of presumably sporadic cases. Recent studies suggest that MI in many cell lines or xenografts derived from sporadic colorectal carcinomas is not attributable to mutations in four known human DNA mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, and hPMS2). Mutational analyses of these four MMR genes in endometrial carcinomas have not been previously reported. We analyzed nine sporadic MI-positive primary endometrial carcinomas for mutations in the above four MMR genes. Mutations were detected in two tumors (in hMSH2), and both of the mutations were acquired somatically. Immunohistochemical staining revealed a lack of expression of hMSH2 protein in the two tumors containing hMSH2 mutations. Our data suggest that mutations in these four known DNA MMR genes are not responsible for MI in the majority of sporadic endometrial carcinomas displaying this phenotype.

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts.

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage repair in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.

Frequency of homozygous deletion at p16/CDKN2 in primary human tumours.

Many tumour types have been reported to have deletion of 9p21 (refs 1-6). A candidate target suppressor gene, p16 (p16INK4a/MTS-1/CDKN2), was recently identified within the commonly deleted region in tumour cell lines. An increasing and sometimes conflicting body of data has accumulated regarding the frequency of homozygous deletion and the importance of p16 in primary tumours. We tested 545 primary tumours by microsatellite analysis with existing and newly cloned markers around the p16 locus. We have now found that small homozygous deletions represent the predominant mechanism of inactivation at 9p21 in bladder tumours and are present in other tumour types, including breast and prostate cancer. Moreover, fine mapping of these deletions implicates a 170 kb minimal region that includes p16 and excludes p15.

p53 inactivation by HPV16 E6 results in increased mutagenesis in human cells.

To study the pathways associated with genomic instability in cancer, we examined UV-induced and spontaneous mutagenesis in clonal cell lines expressing human papillomavirus (HPV) proteins, either high-risk (HPV16) E6 or E7 or low-risk (HPV11) E6, in comparison to the parental RKO cells, a colon carcinoma cell line expressing only normal p53. High-risk E6 and E7 bind and functionally inactivate tumor suppressor proteins p53 and Rb, respectively, and both disrupt the G1 arrest in response to DNA damage. Low-risk HPV E6 proteins bind p53 with much lower affinity than high-risk E6 and fail to mediate p53 degradation or to disrupt the G1 checkpoint. We found that cells expressing HPV16 E6 had reduced survival and increased mutagenesis at the hprt locus when treated with low doses of UV. However, this analysis was complicated by the unexpected observation of a very high background of spontaneous mutagenesis in the unirradiated cells expressing the HPV16 E6 gene. Fluctuation analysis revealed a 5-fold elevated mutation rate in the cells expressing HPV16 E6. HPV11 E6 conferred a 2-fold elevation in the mutation rate, but HPV16 E7 had no effect. The increased spontaneous mutagenesis, therefore, appeared to be mediated by p53 inactivation and to be independent of Rb (which acts downstream of p53 in the G1 arrest pathway following DNA damage). Taken together, these findings suggest that the effect of p53 inactivation on spontaneous mutagenesis is manifested at the level of DNA repair, recombination, or coupling of transcription with one of these processes instead of by an alteration in G1 arrest.