Cytogenetic characterization and H-ras associated transformation of immortalized human mammary epithelial cells.

INTRODUCTION: Immortalization is a key step in malignant transformation, but immortalization alone is insufficient for transformation. Human mammary epithelial cell (HMEC) transformation is a complex process that requires additional genetic changes beyond immortalization and can be accomplished in vitro by accumulation of genetic changes and expression of H-ras. METHODS: HMEC were immortalized by serial passaging and transduction with the catalytic subunit of the human telomerase gene (hTERT). The immortalized cells were passaged in vitro and studied by a combination of G-banding and Spectral Karyotyping (SKY). H-ras transduced, hTERT immortalized cells were cloned in soft agar and injected into nude mice. Extensive analysis was performed on the tumors that developed in nude mice, including immunohistochemistry and western blotting. RESULTS: Immortal HMEC alone were not tumorigenic in gamma-irradiated nude mice and could not grow in soft agar. Late passage hTERT immortalized HMEC from a donor transduced with a retroviral vector containing the mutant, autoactive, human H-ras61L gene acquired anchorage independent growth properties and the capacity for tumorigenic growth in vivo. The tumors that developed in the nude mice were poorly differentiated epithelial carcinomas that continued to overexpress ras. These cells were resistant to doxorubicin mediated G1/S phase arrest but were sensitive to treatment with a farnesyltransferase inhibitor. CONCLUSION: Some of the cytogenetic changes are similar to what is observed in premalignant and malignant breast lesions. Despite these changes, late passage immortal HMEC are not tumorigenic and could only be transformed with overexpression of a mutant H-ras oncogene.

Penile cancer: importance of circumcision, human papillomavirus and smoking in in situ and invasive disease.

Few population-based case-control studies have assessed etiologic factors for penile cancer. Past infection with high-risk human papillomavirus (HPV) is a known risk factor for penile cancer; however, few previous studies have related the HPV DNA status of the tumor to potential demographic and behavioral risk factors for the disease or evaluated whether in situ and invasive penile cancer share risk factors. Little information is available on the role and timing of circumcision in the etiology of penile cancer. We conducted a population-based case-control study in western Washington state that included 137 men diagnosed with in situ (n = 75) or invasive (n = 62) penile cancer between January 1, 1979, and December 31, 1998, and 671 control men identified through random digit dialing. Cases and controls were interviewed in person and provided peripheral blood samples. Case and control blood samples were tested for antibodies to HPV16 and HSV-2, and tumor specimens from cases were tested for HPV DNA. Men not circumcised during childhood were at increased risk of invasive (OR = 2.3, 95% CI 1.3-4.1) but not in situ (OR = 1.1, 95% CI 0.6-1.8) penile cancer. Approximately 35% of men with penile cancer who had not been circumcised in childhood reported a history of phimosis compared to 7.6% of controls (OR = 7.4, 95% CI 3.7-15.0). Penile conditions such as tear, rash and injury were associated with increased risk of disease. Among men not circumcised in childhood, phimosis was strongly associated with development of invasive penile cancer (OR = 11.4, 95% CI 5.0-25.9). When we restricted our analysis to men who did not have phimosis, the risk of invasive penile cancer associated with not having been circumcised in childhood was not elevated (OR = 0.5, 95% CI 0.1-2.5). Cigarette smoking was associated with a 4.5-fold risk (95% CI 2.0-10.1) of invasive penile cancer. HPV DNA was detected in 79.8% of tumor specimens, and 69.1% of tumors were HPV16-positive. The proportion of HPV DNA-positive tumors did not vary by any risk factors evaluated. Many risk factors were common for both in situ and invasive disease. However, 3 factors that did not increase the risk for in situ cancer proved significant risk factors for invasive penile cancer: lack of circumcision during childhood, phimosis and cigarette smoking. The high percentage of HPV DNA-positive tumors in our study is consistent with a strong association between HPV infection and the development of penile cancer regardless of circumcision status. Circumcision in early childhood may help prevent penile cancer by eliminating phimosis, a significant risk factor for the disease.

Human papillomavirus, smoking, and sexual practices in the etiology of anal cancer.

BACKGROUND: The incidence of anal cancer has increased among both men (160%) and women (78%) from 1973 to 2000 in the U.S. The authors conducted a population-based case-control study of anal cancer to examine factors that may account for this increase. METHODS: Men (n = 119 patients) and women (n = 187 patients) who were diagnosed with anal cancer between 1986 and 1998 in the Seattle area were ascertained through the local Surveillance, Epidemiology, and End Results registry. Control participants (n = 1700) were ascertained through random-digit telephone dialing. Participants were interviewed in person and provided blood samples. Archival tumor tissue was tested for human papilloma virus (HPV) DNA, and serum samples were tested for HPV type 16 (HPV-16). RESULTS: Overall, 88% of tumors (all histologies) in the study were found to be positive for HPV. HPV-16 was the most frequent HPV type detected (73% of all tumors), followed by HPV-18 (6.9%), regardless of gender. However, 97.7% of tumors from men who were not exclusively heterosexual contained HPV DNA. The risk of anal cancer increased among men (odds ratio [OR], 5.3; 95% confidence interval [95% CI], 2.4-12.0) and women (OR, 11.0; 95% CI, 5.5-22.1) who had > or = 15 sexual partners during their lifetime. Among men who were not exclusively heterosexual and women, receptive anal intercourse was related strongly to the risk of anal cancer (OR, 6.8 [95% CI, 1.4-33.8] and OR, 2.2 [95% CI, 1.4-3.3], respectively). Current smokers among men and women were at particularly high risk for anal cancer, independent of age and other risk factors (OR, 3.9 [95% CI, 1.9-8.0] and OR, 3.8 [95% CI, 2.4-6.2], respectively). CONCLUSIONS: The high proportion of tumors with detectable HPV suggests that infection with HPV is a necessary cause of anal cancer, similar to that of cervical cancer. Increases in the prevalence of exposures, such as cigarette smoking, anal intercourse, HPV infection, and the number of lifetime sexual partners, may account for the increasing incidence of anal cancer in men and women.

Telomere erosion and chromosomal instability in cells expressing the HPV oncogene 16E6.

Progression to advanced-stage cervical carcinomas is characterized by a recurrent pattern of chromosomal rearrangements. Structural chromosome rearrangements are generated through the fusion of broken chromosome ends. These chromosome breaks may be induced by mutagenic agents such as ionizing radiation, or chromosome ends may be exposed through extensive telomere shortening. The human papilloma virus oncogene 16E6 induces telomerase activity in human keratinocytes, a model system for cervical tumor formation. The present study explores the relationship between 16E6 expression, telomerase activity, and chromosomal instability. We show that the frequency of anaphase bridges is dependent on the level of telomerase activity in 16E6/E7-expressing clones, and is the result of telomere shortening. High frequencies of anaphase bridges, associated with low telomerase activity, correlate with increased chromosome instability. Anaphase bridge formation is also associated with the presence of micronuclei, which are shown to contain unstable chromosomes frequently involved in rearrangements. As anaphase bridges are observed in both high and low telomerase 16E6/E7 clones, but not in hTERT-expressing control clones, expression of 16E6 in these immortalized clones is not sufficient to stabilize shortened telomeres completely. We suggest a model in which HPV-induced tumorigenesis may be dependent on persistent bridge-breakage-fusion cycles that allow for continued genomic rearrangements.

Production of spindle cell carcinoma by transduction of H-Ras 61L into immortalized human mammary epithelial cells.

Human mammary epithelial cells (HMEC) were immortalized by serial passaging through senescence (M0) and subsequent transduction with the catalytic subunit of the human telomerase gene (hTERT). These cells acquired multiple non-random cytogenetic abnormalities with lengthy passaging in vitro, but are still not tumorigenic in irradiated nude mice and cannot grow in soft agar. Transduction, of late passage immortal HMEC from a single donor, with a retroviral vector containing the mutant autoactive H-Ras 61L gene, enabled immortal HMEC to acquire anchorage independent growth properties. Three colonies were picked and all three were found to be tumorigenic. One colony exclusively produced epithelial tumors in nude mice, but the other two colonies gave rise exclusively to malignancies in which the cells displayed a spindle morphology. In this paper we describe the characteristics of the tumors arising from one of these 'spindle colonies'. These tumors were strongly positive for vimentin staining and virtually negative for pan-cytokeratin staining, on immunohistochemistry. Cytogenetic analysis of the cells derived from these tumors confirmed that they were derived from the original cultured, immortalized mammary cells. We conclude that the HMEC have undergone metaplastic transformation due to the high levels of H-Ras 61L and telomerase activity that they display, and the derived tumors are best described as spindle cell carcinomas.

Human leukocyte antigen class II and cervical cancer risk: a population-based study.

The critical role of the human leukocyte antigen (HLA) system in presenting peptides to antigen-specific T cell receptors may explain why only some human papillomavirus (HPV)-infected women progress to cervical cancer. HLA class II DRB1 and DQB1 genes were examined in 315 women with invasive squamous cell cervical cancer (SCC) and 381 control subjects. Increased risks of SCC were associated with DRB1*1001, DRB1*1101, and DQB1*0301, and decreased risks were associated with DRB1*0301 and DRB1*13. Of squamous cell tumors, those containing HPV-16 were different from those not containing HPV-16 for 3 alleles: DRB1*0401, DRB1*07, and DQB1*06. Increased risks of SCC were associated with DRB1*0401-DQB1*0301 (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.1-2.7) and DRB1*1101-DQB1*0301 (OR, 2.5; 95% CI, 1.4-4.5), and decreased risks were associated with DRB1*0301-DQB1*02 (OR, 0.7; 95% CI, 0.5-1.0) and DRB1*13-DQB1*06 (OR, 0.6; 95% CI, 0.4-0.9) haplotypes. These results add to the evidence that certain HLA class II alleles or allele combinations, or genes linked to them, make some women more susceptible to SCC.

The human papillomavirus type 16 E6 oncogene induces premature mitotic chromosome segregation.

Expression of the human papillomavirus type 16 E6 and E7 oncogenes initiates and maintains abnormal cell replication, by interacting with the p53 and retinoblastoma (Rb) gene products. Subsequent changes in host cell gene expression, as a consequence of genetic instability, can result in progression to invasive carcinoma. In addition to previously described effects of these viral oncogenes on centrosome synthesis, primarily associated with the expression of E7, the results described herein demonstrate that the E6 oncogene can induce premature chromosome segregation in human cells.

A population-based study of squamous cell vaginal cancer: HPV and cofactors.

BACKGROUND: Little is known about the etiology of in situ or invasive squamous cell cancer of the vagina. It is thought that some vaginal cancers may have the same etiology as cervical cancer. It is also not known whether in situ and invasive vaginal cancer share the same etiologic factors. We conducted a study to evaluate risk factors for in situ and invasive vaginal cancer and their potential relationship to prior exposure to human papillomaviruses (HPV). METHODS: A population-based case-control study included 156 women with squamous cell in situ or invasive vaginal cancer diagnosed between January 1981 and June 1998 and 2041 control women identified through random-digit dialing in western Washington state. Cases and controls were interviewed in person and provided blood samples; archival tumor tissue was retrieved for cases. Blood samples were tested for antibodies to HPV, and tumor tissue was tested for HPV DNA. RESULTS: Women with vaginal cancer were more likely to have five or more lifetime sexual partners (OR = 3.1, 95% CI 1.9 to 4.9), to have an early age at first intercourse (<17 years OR = 2.0, 95% CI 1.2 to 3.5), and to be current smokers at diagnosis (OR = 2.1, 95% CI 1.4 to 3.1) than control women. Approximately 30% of all cases had been treated for a prior anogenital tumor, most often of the cervix. Prior hysterectomy was a risk factor only among women who had no history of prior anogenital cancer (OR = 3.9 95% CI 2.5 to 6.1). Antibodies to HPV16 L1 were strongly related to risk of vaginal cancer (OR = 4.3, 95% CI 3.0 to 6.2). We detected HPV DNA in tumor blocks from over 80% of the patients with in situ and 60% of the patients with invasive cancers. CONCLUSIONS: In situ and invasive vaginal neoplasia have many of the same risk factors as cervical cancer, including a strong relationship to HPV infection. Women who have been treated for a prior anogenital cancer, particularly of the cervix, have a high relative risk, although low absolute risk, of being diagnosed with vaginal cancer.