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Biomedical subjects

D A Galloway

Publications and source records attributed to D A Galloway.

At least 55 records · Page 3Linked to original sources
PubMed1996

Human papillomavirus type 16 E7 alleviates a proliferation block in early passage human mammary epithelial cells.

Human mammary epithelial cells (HMEC) isolated from reduction mammoplasty tissue are proliferative for several passages but then enter a period termed ¿selection' or M0 during which the majority of the cells become larger, flattened, and less proliferative. Early passage HMEC (prior to M0) were transduced with human papillomavirus type-16 E6, E7, or E6/E7 by using recombinant retroviral vectors. E7 alone or E6/E7, but not E6 alone, alleviated the M0 proliferation block, suggesting a possible role for Rb or Rb-related proteins, but not p53, in M0. In addition, cells in M0 did not have increased levels of p53 or p21 proteins, further indicating that induction of these proteins is not required for the M0 proliferation block. Early passage cells contained Rb which was primarily hyperphosphorylated while cells in M0 contained Rb protein which was predominantly underphosphorylated. Cells in M0 accumulated in G1 or B0 and expressed reduced levels of cyclin A and CDK2 proteins.

Animals↗
PubMed1996

Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents.

Many tumor types have p53 and/or RB mutations, and it is unclear what role the mutations of these tumor suppressor genes have on the efficacy of chemotherapeutic agents. The effect of p53 and RB inactivation on sensitivity to chemotherapeutic drugs was examined using a model system in which p53 or RB was inactivated in normal human foreskin fibroblasts (HFFs) by acute expression of human papillomavirus (HPV) 16E6 or 16E7. Cytotoxicity assays showed that HFFs expressing HPV 16E6 were 6- to 9-fold more sensitive to the DNA crosslinkers cisplatin and carboplatin and 7.8- to 11.5-fold more sensitive to the tubulin polymerizing agent paclitaxel than were LXSN-expressing cells. Analysis of mouse embryonal fibroblasts lacking p53 (p53-/-) compared with mouse embryonal fibroblasts homozygous (p53+/+) and heterozygous (p53+/-) for wild-type p53 confirmed the role of p53 in the enhanced sensitivity to cisplatin. Treatment with the alkylating agents melphalan and nitrogen mustard resulted in 3.8- to 7.3-fold greater sensitivity in HPV 16E6- or 16E7-expressing cells compared with LXSN-expressing cells. Enhanced sensitivity to cisplatin in cells lacking p53 function was explored by examination of its effects on cell cycle progression after exposure. When treated with cisplatin, HFFs expressing 16E6 showed delayed progression through S phase relative to HFFs expressing LXSN. The delay in S phase progression was coincident with the induction of p53 protein levels in LXSN-containing HFFs, suggesting a role for p53 in DNA repair of cisplatin-induced damage. These results indicate that the inactivation of p53 in the absence of other genetic alterations leads to enhanced sensitivity to multiple chemotherapeutic agents rather than to increased resistance.

Animals↗
PubMed1996

The disruption of cell cycle checkpoints by papillomavirus oncoproteins contributes to anogenital neoplasia.

Human cancer are characterized by the failure of cell cycle checkpoints resulting in genetic instability. Human papillomaviruses contribute to the development of anogenital malignancies because the E6 and R7 oncoproteins from high risk HPV types are able to disrupt the integrity of these checkpoints. HPV 16 E7 prevents suprabasal cells from exiting the cell cycle, thus increasing the pool of replicating cells that are available for additional 'hits'. Cells that suffer DNA of chromosome damage are not eliminated because E6 and E7 are able to bypass G1 and G2 damage-induced checkpoints. The activation, or inactivation, of additional cellular genes required for invasion and metastasis may not be a direct consequence of the E6/E7 oncoproteins.

Anal Gland Neoplasms↗
PubMed1996

Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis.

The anticancer agent paclitaxel (Taxol) stabilizes tubulin polymerization resulting in arrest in mitosis and apoptotic cell death. Normal human fibroblasts depleted of functional p53 by SV40 T antigen or HPV-16 E6, and primary embryo fibroblasts from p53 null mice showed seven- to ninefold increased cytotoxicity by paclitaxel. Reduced levels of p53 correlated with increased G2/M phase arrest, micronucleation, and p53-independent paclitaxel-induced apoptosis. Surviving cells with intact p53 progressed through mitosis and transiently accumulated in the subsequent G1 phase, coincident with increased p53 and p21cip1,waf1 protein levels. These results are in contrast to studies linking p53 loss with resistance to DNA damaging anticancer agents.

Animals↗
PubMed1996

The natural history of human papillomavirus type 16 capsid antibodies among a cohort of university women.

To study the temporal relationship between serum antibody response and human papillomavirus type 16 (HPV-16) infection, a cohort of 325 university women were scheduled for examinations at 4-month intervals. At every examination, interviews were completed, cells were obtained for polymerase chain reaction-based testing and for Pap screening, and serum was obtained for testing with a HPV-16 capsid-capture ELISA. Seroreactivity was associated with detection of HPV-16 DNA and with increased numbers of sex partners. The median time to seroconversion was 8.3 months among women with incident HPV-16 infections. Within 16 months following HPV-16 DNA detection, 93.7% of women with prevalent and 67.1% of women with incident infections seroconverted. After seroconversion, antibody responses were maintained during follow-up among HPV-16 DNA-positive women. Women who seroconverted were 5.7 times (95% confidence interval = 2.4-13.4) more likely to have squamous intraepithelial lesions associated with the detection of HPV-16 DNA than were women who did not seroconvert.

Adolescent↗
PubMed1996

Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.

Cell Cycle↗
PubMed1996

Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16.

Pancreatic cancer is one of the most lethal cancers in humans. The majority of these cancers arise from the pancreatic duct epithelium. Research into the pathogenesis of pancreatic carcinoma has largely relied on animal models. In vitro models of pancreatic carcinogenesis using propagable cultured epithelial cells derived from the pancreatic ducts of rats and hamsters have been described. A human model, however, has been nonexistent due to the unavailability of propagable cultured duct epithelial cells derived from normal human pancreas. We report here a reproducible method for the long-term culture of pancreatic duct epithelial cells derived from normal and benign adult human pancreata by infection with a retrovirus containing the E6 and E7 genes of the human papilloma virus 16. One of these cell lines has become immortal and has propagated continuously for more than 20 passages. They remain anchorage dependent in their growth and nontumorigenic in nude mice. These cell lines and the methodology described here to establish them may provide new avenues for in vitro studies of the roles played by duct epithelium in human pancreatic diseases and cancers.

Aged↗
PubMed1996

The relationship of human papillomavirus-related cervical tumors to cigarette smoking, oral contraceptive use, and prior herpes simplex virus type 2 infection.

It has now been established that infection with human papillomavirus (HPV) is necessary for the development of most cervical cancers. HPV is not sufficient for the development of cancer. Other exposures or host factors are necessary for cancer to occur. As part of an ongoing, population-based case-control study of invasive cervical cancer, we investigated the role of cigarette smoking, oral contraceptive (OC) use, and herpes simplex virus type 2 (HSV-2) as potential cofactors with HPV in the development of cervical cancer. Residents of three counties in western Washington State who were diagnosed with invasive squamous cell cervical cancer (n = 314) from January 1986 through December 1992 were interviewed about their sexual, reproductive, contraceptive, and cigarette smoking histories. Similar information was obtained from control women identified through random digit dialing (n = 672). The sera from 206 cases and 522 controls were tested for both HPV 16 capsid antibodies and HSV-2 antibodies. PCR was used to test paraffin-embedded tumor tissues for the presence of HPV DNA types 6, 16, 18, 31, 33, 35, and 39. Women with cervical cancer were more likely to be current smokers at diagnosis than population controls [relative risk (RR), 2.5; 95% confidence interval (CI), 1.8-3.4]. The risk associated with smoking was present to a similar extent among women positive and negative for HPV as measured by HPV 16 capsid antibodies and HPV DNA in the tumor tissue (cases). OC use was only important if first use was at an early age, particularly ages < or = 17 years (RR, 2.3; 95% CI, 1.4-3.8). There was only a slight risk for cervical cancer associated with antibodies to HSV-2 (RR, 1.2; 95% CI, 0.9-1.7). However, when we stratified by markers of HPV exposure, we found a significant increase in risk associated with HSV-2 among women negative for HPV 16 antibodies (RR, 2.0; 95% CI, 1.3-3.0), which was strengthened when we confined our analysis to cases whose tumors were HPV DNA negative (RR, 3.6; 95% CI, 1.6-8.0). There was no indication that cigarette smoking, OC use, or HSV-2 infection influence the ability of HPV infection to cause invasive cervical cancer. OC use may only be important in the etiology of invasive squamous cell cervical tumors if the use occurs at a critical time in the development of a woman's reproductive tract, at ages < or = 17 years. The majority of risk associated with HSV-2 was confined to HPV-negative tumors, indicating a possible separate pathway to disease that may account for 5-10% of invasive cervical cancers.

Adolescent↗
PubMed1995

p21CIP1 is not required for the early G2 checkpoint response to ionizing radiation.

We have previously reported that the immediate G2 checkpoint delay of normal human fibroblasts in response to ionizing radiation is correlated with inhibition of p34CDC2/cyclin B kinase activity. Here, we observed increased amounts of the cyclin-dependent protein kinase inhibitor p21CIP1 associated with p34CDC2/cyclin B protein complexes from irradiated normal human fibroblasts. Since wild-type p53 function is not required for the early G2 checkpoint response to ionizing radiation, we investigated whether a p53-independent induction of p21CIP1 was required for the G2 checkpoint. Early passage human fibroblasts expressing the E6 oncoprotein of human papilloma virus-type 16 (NHF4 E6) were analyzed. It has been demonstrated earlier than inactivation of wild-type p53 function in these cells by E6 protein does not alter their intact early G2 checkpoint response to gamma-rays. p21CIP1 was found to be undetectable in p34CDC2/cyclin B protein complexes and in total extracts from the E6-expressing cells, with or without exposure to ionizing radiation. These data indicate that p21CIP1 is not required for the immediate G2 checkpoint response and is not induced by a p53-independent pathway in G2 phase following exposure to gamma-rays.

Cell Cycle↗
PubMed1995

Abrogation of the G2 checkpoint results in differential radiosensitization of G1 checkpoint-deficient and G1 checkpoint-competent cells.

We have examined the effect of abrogation of the G2 checkpoint on the radiosensitivity of G1 checkpoint-proficient and G1 checkpoint-deficient cells. A549 human lung adenocarcinoma cells were transduced with the E6 oncogene of the human papillomavirus type 16 to eliminate their radiation-induced G1 arrest. These E6+ cells exhibited a dose-dependent increase in radiation resistance compared to control A549 cells transduced with the vector alone. Treatment (96 h) with 2 mM caffeine resulted in an abrogation of the cellular G2 checkpoint in both E6+ and control cells and a differential radiosensitizing effect on the two cell lines such that the E6+ clones and the vector controls became equally radiosensitive. These data show that human tumors which are radioresistant due to the loss of the p53-mediated G1 checkpoint can be made radiosensitive by abrogation of the G2 checkpoint. The implications of these results for cancer therapy are discussed.

Adenocarcinoma↗
PubMed1995

Defective G2 checkpoint function in cells from individuals with familial cancer syndromes.

The early events in the G2 checkpoint response to ionizing radiation (IR) were analyzed in diploid normal human fibroblasts (NHFs) and fibroblasts from patients with two heritable cancer syndromes. Exposure to gamma-radiation of asynchronously growing NHFs resulted in a rapid reduction in the number of cells in mitosis (G2 delay) and was accompanied by a quantitatively similar reduction in the p34CDC2/cyclin B in vitro histone H1 kinase activity as compared with sham-treated controls. This G2 delay was strong by 1 h following exposure to IR, maximal by 2 h, and was accompanied by an accumulation of tyrosine-phosphorylated p34CDC2 molecules. In contrast, fibroblasts from individuals with ataxia telangiectasia displayed significantly less reduction of the mitotic index or histone H1 kinase activity after IR. Low passage fibroblasts from individuals with Li-Fraumeni syndrome having one wild-type and one mutated p53 allele were similar to NHFs in their immediate G2 checkpoint response to IR, as were NHFs expressing the human papilloma virus type 16 E6 gene product (functionally inactivating p53) and low passage cells from p53-deficient mouse embryos. However, the p53-deficient fibroblasts were genomically unstable and became defective in their early G2 checkpoint response to IR. Furthermore, immortal Li-Fraumeni syndrome fibroblasts lacking wild-type p53 displayed an attenuated G2 checkpoint response. These results link the early events in G2 checkpoint response to IR in NHFs with a rapid inhibition of p34CDC2/cyclin B protein kinase activity and demonstrate that while not required for this immediate G2 delay, lack of p53 can lead to subsequent genetic alterations that result in defective G2 checkpoint function.

Ataxia Telangiectasia↗
PubMed1995

Immunization of mice with HPV vaccinia virus recombinants generates serum IgG, IgM, and mucosal IgA antibodies.

To assess the utility of vaccinia virus recombinants in the development of an immune response against HPV capsid antigens, 5-week-old C57B16 female mice were administered either purified HPV 1 capsids produced by a vaccinia virus recombinant or the recombinant vaccinia virus itself. Animals were boosted at Week 4 with either agent. Mice developed a serum IgG antibody response in all the administration protocols that was directed mainly against native L1 epitopes. Mice injected initially with the vaccinia virus recombinant and boosted with purified capsids had a higher titer antibody response (P = 0.024) with more mice responding to a greater extent. All mice produced a serum IgM response that preceded the IgG response by approximately 2 weeks and lasted 1-3 weeks. The IgM response was directed against native L1 epitopes. Although no serum IgA was detected, IgA could be detected in vaginal secretions of mice that were immunized or boosted with the vaccinia virus vector. These results indicate that an extensive humoral immune response to HPV can be elicited using vaccinia virus recombinants.

Animals↗
PubMed1995

Analysis of human papillomavirus type 16 variants indicates establishment of persistent infection.

Sequence differences in the noncoding region of the human papillomavirus type 16 (HPV-16) genome were displayed using single-stranded conformational polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified material. Two variants accounted for 50%-70% of all HPV-16 variants from 3 cohorts in Seattle. Seventy subjects who were repeatedly HPV-16 DNA-positive over 2-8 4-monthly visits showed an identical SSCP pattern at every visit. Only 10%-20% of the specimens showed evidence of infection by multiple variants when assessed by SSCP. However, cloning and sequencing of the PCR products revealed a substantially higher proportion of specimens with > 1 variant. Sequencing many clones from each specimen confirmed that 1 major variant seemed to predominate over time, whereas minor variants appeared more transient. These results suggest that HPV-16 establishes a persistent infection in which a single variant predominates: coinfection with addition HPV-16 variants results in a minor population of HPV-16 genomes.

Base Sequence↗
PubMed1995

Serological and molecular evidence of rhesus papillomavirus type 1 infections in tissues from geographically distinct institutions.

We have previously demonstrated the presence of rhesus monkey papillomavirus type 1 (RhPV-1), from molecular and pathological evidence, in a mating group within a single institution. We have now also obtained a number of fresh or archival tissues of rhesus monkeys from other geographically distinct institutions. Using PCR amplification, we observed two animals from one of these institutions and five animals from another which demonstrated RhPV-1 DNA sequences. In addition we molecularly cloned the E7, E2, E4, L2 and L1 genes of RhPV-1 into bacterial expression vectors. The fusion gene products were used to test for serological response to RhPV-1 antigens by Western blot analysis. Responses were observed in up to 52% of the animals tested. While some serologically positive animals were also RhPV-1 DNA-positive, most were not.

Animals↗
PubMed1994

Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7.

Cellular tumor suppressors p53 and Rb play an important role in controlling cell proliferation. Inactivation of these tumor suppressor proteins can occur by gene mutation or by association with oncoproteins from the small DNA tumor viruses. One function of p53 is in regulating cell cycle check-point control after DNA damage. To dissect the pathways by which p53 and Rb may act, the E6 and E7 oncogenes of human papillomavirus (HPV) types 6 and 16 were introduced into primary human epithelial cells by retroviral transfer vector, and cells were assayed for growth arrest after DNA damage induced by actinomycin D. The E6 or E7 oncogenes from the low-risk HPV6 had no affect on growth arrest, p53 protein levels increased, Rb protein levels decreased, and Rb was predominantly in the hypophosphorylated state similar to vector-infected cells. Either the E6 or the E7 oncogene from the high-risk HPV16 abrogated growth arrest. Cells expressing HPV16 E6 (16E6) were severely reduced in p53 protein levels that did not increase detectably after DNA damage, Rb protein levels did not decrease, and hyperphosphorylated Rb was present. After DNA damage in cells expressing 16E7 p53 levels increased, and Rb protein levels decreased; however, Rb was predominantly in the hyperphosphorylated state. Even though p53 protein levels increased in response to DNA damage in cells expressing 16E7, G1 growth arrest was bypassed. This suggests that the circuitry controlling the growth arrest signal after DNA damage may be interconnected between the p53 and Rb pathways.

Blotting, Western↗
PubMed1994

Elevated wild-type p53 protein levels in human epithelial cell lines immortalized by the human papillomavirus type 16 E7 gene.

The role tumor suppressors p53 and retinoblastoma (RB) play in the transformation process has become central to understanding the pathogenesis of DNA tumor viruses. The two oncoproteins of human papillomavirus (HPV)-16, E6 and E7, bind to p53 and RB, respectively, thus inactivating the function of these tumor suppressor genes. Immortalization of primary human foreskin epithelial cells by HPV requires expression of the E7 protein, and the E6 protein greatly enhances the immortalization frequency. Two of three cell lines immortalized by the HPV-16 E7 oncoprotein expressed wild-type p53 and only one of the three cell lines had acquired a p53 mutation and loss of heterozygosity at 17p during the immortalization process. All three E7-immortalized lines contained higher steady-state levels of p53 protein. Mutation of the p53 gene is not required for immortalization in the absence of the HPV-16 E6 inactivation of the p53 protein, and 16E7 expression leads to the stabilization of wild-type p53.

Base Sequence↗
PubMed1994

Use of HPV 1 capsids produced by recombinant vaccinia viruses in an ELISA to detect serum antibodies in people with foot warts.

A sandwich ELISA was developed to detect HPV antibodies using HPV 1 capsids that were purified from recombinant vaccinia virus-infected cells and a monoclonal antibody to the HPV 1 L1 protein. Sera from 91 college-aged women who had been previously screened for HPV 1 antibodies by immune precipitation of capsid proteins were tested by ELISA. A cutoff point was established independently of other criteria based on the assumption that the ELISA values came from a mixture of two Normal distributions representing seropositive and seronegative individuals. It was found that the data fit this model best when the natural log of the ELISA (+0.5 to make all of the values positive) was used. Positive sera were shown to react with a conformational epitope(s) on the L1 protein. In the population reporting foot warts, 16 of 18 (89%) had ELISA values above the cutoff. This compared to 38 of 73 (53%) positives in the population reporting no history of foot warts. The odds ratio for the association of the ELISA reactivity with foot warts was 7.23 (95% CI 1.53, 69.4; P < 0.01). There was no significant association between the ELISA reactivity and wart infections reported at other sites. The average of the log ELISA values for individuals never reporting foot warts was -0.223 (SD 0.468), whereas the average value for individuals reporting foot warts within 10 years was 0.191 (SD 0.450) (P = 0.001). There was a negative correlation between the magnitude of ELISA reactivity and the time elapsed since the last appearance of foot warts. This apparent loss of seroreactivity over time may indicate that HPV 1 is usually eliminated from the host after infection or that inadequate levels of HPV 1 capsid antigen are produced during latent foot warts to maintain antibody levels.

Adolescent↗