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D A Galloway

Publications and source records attributed to D A Galloway.

At least 37 records · Page 2Linked to original sources
PubMed1998

Type specific and genotype cross reactive B epitopes of the L1 protein of HPV16 defined by a panel of monoclonal antibodies.

Mouse monoclonal antibodies (mAbs) were raised against the major capsid protein, L1, of human papillomavirus type 16 (HPV16), produced in Escherichia coli with the expression plasmid pTrcL1. Epitope specificity could be assigned to 11 of these 12 antibodies using a series of linear peptides and fusion proteins from HPV16. One mAb (MC53) recognized a novel linear epitope that appears to be unique to the HPV16 genotype. A further 11 mAbs were characterized as recognizing novel and previously defined linear and conformational epitopes shared among more than one HPV genotype. The apparently genotype specific mAb could be useful for the development of diagnostic tests for vegetative virus infection in clinical specimens.

Amino Acid Sequence↗
PubMed1998

Low risk of perinatal transmission of human papillomavirus: results from a prospective cohort study.

OBJECTIVE: Our purpose was to evaluate the risk of perinatal transmission of human papillomavirus. STUDY DESIGN: Pregnant women were evaluated at <20 weeks' and between 34 and 36 weeks' gestation for genital human papillomavirus by clinical and colposcopic examination and by polymerase chain reaction. Their 151 infants were evaluated at birth, 6 weeks, and 6, 12, 18, 24, and 36 months of age for detection of human papillomavirus deoxyribonucleic acid by polymerase chain reaction on samples from the mouth, external genitalia, and anus. Polymerase chain reaction was performed with human papillomavirus L1 consensus primers and hybridization to human papillomavirus types 6, 11, 16, 18, 31, 33, 35, 39, and 45 and to a generic probe. RESULTS: During pregnancy 112 (74%) of 151 women had historic, clinical, or deoxyribonucleic acid evidence of genital human papillomavirus infection. At 479 infant visits, human papillomavirus deoxyribonucleic acid was detected from only five (1.5%) of the 335 genital, four (1.2%) of the 324 anal, and none of the 372 oral or nasopharyngeal specimens. A positively reacting specimen was obtained from three (4%) of 80 infants born to women with human papillomavirus deoxyribonucleic acid detected at 34 weeks' gestation and from five (8%) of 63 born to women without human papillomavirus deoxyribonucleic acid (p = 0.47). All positive results in the infants were positive only with the generic probe and were preceded or followed by negatively reacting specimens. No clinical manifestations of human papillomavirus infection were detected in any infant. CONCLUSIONS: The isolated detection of unclassified human papillomavirus types from infants at only single visits may represent low-level genital or nongenital human papillomavirus or may represent contamination. Although perinatal transmission of human papillomavirus is not ruled out by these data, the upper 95% confidence interval for detection of perinatal transmission from women with any evidence of genital human papillomavirus was only 2.8%.

Adolescent↗
PubMed1998

Inhibiting CDK inhibitors: new lessons from DNA tumor viruses.

The ability of viral oncoproteins to inactivate the retinoblastoma and p53 tumor suppressors provides mechanisms for bypassing the normal inhibitory activities of cyclin-dependent-kinase inhibitors (CKIs). Recent studies point to novel mechanisms for inhibiting the activities of the CKIs p21CIP1 and p27KIP1 activity. Such mechanisms involve the binding of viral oncoproteins or other proteins to these CKIs and suggest a model in which the cell-cycle activities of p21CIP1 are coordinated through protein-protein interactions.

Cell Cycle↗
PubMed1998

Genital human papillomavirus infection in women who have sex with women.

Genital infection with human papillomavirus (HPV), as determined by polymerase chain reaction detection of HPV DNA and prevalence of HPV-6 and -16 serum antibodies, was investigated in 149 women who were sexually active with women. By use of HPV L1 consensus primers and hybridization to types 6/11, 16, 18, 31/33/35/39, and 45 and a generic probe, HPV DNA was detected in 30% of subjects; of these, 20% had type 31/33/35/39, 18% had type 16, and 2% had type 6/11. Of 21 subjects reporting no prior sex with men, HPV DNA was detected in 19% and squamous intraepithelial lesions in 14%. By capture ELISA with HPV-6 and -16 L1 capsids, 47% of subjects were seropositive for HPV-16 and 62% for HPV-6. Current smoking was associated with detectable HPV DNA. Genital HPV infection and squamous intraepithelial lesions are common among women who are sexually active with women and occur among those who have not had sex with men.

Adult↗
PubMed1998

Disruption of the G1/S transition in human papillomavirus type 16 E7-expressing human cells is associated with altered regulation of cyclin E.

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F.

Cells, Cultured↗
PubMed1998

Inactivation of p16 in human mammary epithelial cells by CpG island methylation.

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.

Breast↗
PubMed1997

Cofactors with human papillomavirus in a population-based study of vulvar cancer.

BACKGROUND: Human papillomavirus (HPV) has been previously associated with vulvar cancer. In a population-based study, we examined whether exposure to HPV, cigarette smoking, or herpes simplex virus 2 (HSV2) increases the risk of this cancer. METHODS: Incident cases of in situ (n = 400) and invasive (n = 110) squamous cell vulvar cancer diagnosed among women living in the Seattle area from 1980 through 1994 were identified. Serum samples were analyzed for antibodies against specific HPV types and HSV2. HPV DNA in tumor tissue was detected by means of the polymerase chain reaction. In most analyses, case subjects were compared with population-based control subjects (n = 1403). Relative risks of developing vulvar cancer were estimated by use of adjusted odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Increased risks of in situ or invasive vulvar cancer were associated with HPV16 seropositivity (ORs = 3.6 [95% CI = 2.6-4.8] and 2.8 [95% CI = 1.7-4.7], respectively), current cigarette smoking (ORs = 6.4 [95% CI = 4.4-9.3] and 3.0 [95% CI = 1.7-5.3], respectively), and HSV2 seropositivity (ORs = 1.9 [95% CI = 1.4-2.6] and 1.5 [95% CI = 0.9-2.6], respectively). When the analysis was restricted to HPV16 DNA-positive tumors (in situ or invasive), the OR associated with HPV16 seropositivity was 4.5 (95% CI = 3.0-6.8). The OR for vulvar cancer was 18.8 (95% CI = 11.9-29.8) among current smokers who were HPV16 seropositive in comparison with never smokers who were HPV16 seronegative. CONCLUSIONS: Current smoking, infection with HPV16, and infection with HSV2 are risk factors for vulvar cancer. Risk appears particularly strong among women who are both current smokers and HPV16 seropositive.

Adult↗
PubMed1997

Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein.

p21 inhibits cyclin-dependent kinase (CDK) activity and proliferating cell nuclear antigen (PCNA)-dependent DNA replication by binding to CDK/cyclin complexes and to PCNA through distinct domains. The human papillomavirus (HPV)-16 E7 oncoprotein (16E7) abrogated a DNA damage-induced cell cycle arrest in vivo, despite high levels of p21. Using cell lysates and purified proteins we show that 16E7 prevented p21 both from inhibiting CDK2/cyclin E activity and PCNA-dependent DNA replication, whereas the nononcogenic HPV-6 E7 had reduced effects. Inactivation of both inhibitory functions of p21 was attained through binding between 16E7 and sequences in the carboxy-terminal end of p21 that overlap with the PCNA-binding site and the second p21 cyclin-binding motif. These data imply that the carboxyl terminus of p21 simultaneously modulates both CDK activity and PCNA-dependent DNA replication and that a single protein, 16E7, can override this modulation to disrupt normal cell cycle control.

Binding Sites↗
PubMed1997

Genomic variation of human papillomavirus type 16 and risk for high grade cervical intraepithelial neoplasia.

BACKGROUND: Epidemiologic studies have demonstrated strong and consistent associations between the detection of human papillomavirus (HPV) type 16 DNA and the risk of cervical intraepithelial neoplasia (CIN) and cervical cancer. However, HPV16 is also the most common type of HPV in the normal population, and only a minority of women with HPV16 infection develop cervical cancer. Studies of genomic heterogeneity in HPV16 have demonstrated the presence of multiple variant forms in all human populations examined to date. It is conceivable that the natural variants of HPV16 in a given population may not have the same biologic behavior. PURPOSE: This study was designed to determine the association between natural variants of HPV16 and the risk of biopsy-confirmed CIN 2 or 3, the most important precancerous lesions of the uterine cervix. METHODS: Prospective studies were conducted among 1) women attending a university and 2) women presenting to a sexually transmitted disease clinic. Subjects were eligible for inclusion in this investigation if the initial cytologic findings did not reveal CIN 2-3 and HPV16 DNA was detected by means of a polymerase chain reaction (PCR)-based method in one or more cervical or vulvovaginal samples. Eligible subjects were followed every 4 months with cervical Pap smears and colposcopic examinations. Women were referred for biopsy if cytology or colposcopy suggested CIN 2-3. Two groups of HPV16 variants, prototype-like and nonprototype-like, were determined by means of single-strand conformation polymorphism (SSCP) analysis of PCR products from the noncoding region of the viral genome. Representative SSCP patterns from HPV16 variants were further characterized by direct DNA sequencing of the PCR products. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated by Cox regression analysis. RESULTS: Prototype-like variants accounted for 79% of the HPV16 detected in university students and 86% of the virus detected in patients presenting to the sexually transmitted disease clinic. CIN 2-3 was confirmed by biopsy in nine of 57 HPV16-positive women attending the university and in 10 of 66 HPV16-positive women presenting to the sexually transmitted disease clinic. Among university students, those with HPV16 nonprototype-like variants were 6.5 (95% CI = 1.6-27.2) times more likely to develop CIN 2-3 than those with prototype-like variants. A similar association was observed among women presenting to the sexually transmitted disease clinic (RR = 4.5; 95% CI = 0.9-23.8). CONCLUSIONS: This study suggests that the risk of developing CIN 2-3 is not the same with all variants of HPV16 and that nonprototype-like variants confer a greater risk compared with prototype-like variants. The important genomic differences underlying this increased risk of CIN 2-3 remain to be determined.

Adolescent↗
PubMed1997

Generation of a human melanocyte cell line by introduction of HPV16 E6 and E7 genes.

Availability of a standard human melanocyte cell line with unlimited growth potential and otherwise normal melanocytic properties will greatly facilitate research in melanocyte biology and in vitro studies on the etiology of pigmentary disorders and melanoma. Using a retroviral vector, E6 and E7 open reading frames of human papilloma virus type 16 (HPV 16) have been introduced into cultured normal human melanocytes. Cells selected by increased resistance to geneticin conveyed by the vector and expressing E6E7 mRNA have been cloned to ensure genetic homogeneity. Since their establishment as primary cells, cloned PIG1 cells have undergone more than twice the amount of population doublings of senescent parental cells. Moreover, in passage numbers when parental cells had become senescent, proliferation of clonal cells was retained at levels exceeding those of normal human melanocytes in third passage by 100%. Further characterization has revealed that the cells remain dependent on tetradecanoyl phorbol 13-acetate (TPA) for growth and do not proliferate in soft agar nor form tumors in nude mice. The antigenic profile of the cells was slightly altered as compared to parental cells, but was incomparable to that of M14 melanoma cells. Importantly, PIG1 cells contain more melanin pigment than parental cells.

Animals↗
PubMed1997

Seroprevalence of human papillomavirus types 6 and 16 capsid antibodies in homosexual men.

Human papillomavirus (HPV) has been implicated in the pathogenesis of anal carcinoma, which is increased in homosexual men. Little is known about the serologic response to HPV in normal or immunosuppressed men; therefore, HIV-infected and -uninfected homosexual men were screened for HPV-6 and -16 capsid antibodies. HIV-infected men had increased HPV DNA detection but did not significantly differ in the prevalence of serum HPV antibodies. HPV-6 DNA detection and the presence of anal warts were significantly correlated with serum antibody overall and in the HIV-infected subgroup. HPV-16 DNA detection was not significantly correlated with serum antibody overall or in either subgroup; however, HIV-infected men with high-grade anal squamous intraepithelial lesions were significantly more likely to have HPV-16 antibodies. HIV-infected men are able to generate an antibody response to HPV, and a lack of serum HPV antibodies cannot explain the increased HPV-associated disease seen in HIV-infected men.

AIDS-Related Opportunistic Infections↗
PubMed1997

Inactivation of G2 checkpoint function and chromosomal destabilization are linked in human fibroblasts expressing human papillomavirus type 16 E6.

Chromosomal stability was linked to G2 checkpoint function in human fibroblasts expressing the human papillomavirus type 16 E6 oncoprotein. Soon after expression of E6, cells displayed an undamaged, diploid karyotype and normal mitotic delay after gamma-irradiation. As the E6-expressing cells aged through their in vitro life span, G2 checkpoint function diminished progressively. After 30-70 population doublings, 60-86% of the E6 cells displayed defective G2 checkpoint response. This attenuation of G2 checkpoint function was also associated with radiation-resistant cyclin B1/CDK1 protein kinase activity. Numerical and structural abnormalities of chromosomes developed in unirradiated E6 cells with kinetics that mirrored the loss of G2 checkpoint function. A significant correlation between inactivation of the G2 checkpoint and acquisition of chromosomal abnormalities was found, suggesting that the G2 checkpoint represents a barrier to genetic instability in cells lacking G1 checkpoint function.

CDC2 Protein Kinase↗
PubMed1996

Preferential radiosensitization of G1 checkpoint-deficient cells by methylxanthines.

PURPOSE: To develop a checkpoint-based strategy for preferential radiosensitization of human tumors with deficient and/or mutant p53. METHODS AND MATERIALS: A549 human lung adenocarcinoma cell lines differing in their expression of the p53 tumor suppressor gene were produced by transduction with the E6 oncogene from human papilloma virus type 16. The cells expressing E6 (E6+) lack a G1 arrest in response to ionizing radiation, are deficient in p53 and p21 expression, and exhibit a fivefold greater clonogenic survival following 10 Gy radiation. RESULTS: Postirradiation incubation with millimolar concentrations of the methylxanthine pentoxifylline (PTX) results in preferential radiosensitization of the E6+ cells compared to the LXSN+ vector transduced controls. There is a threefold sensitization of the LXSN+ cells and a 15-fold sensitization of the E6+ cells, which results in equal clonogenic survival of the two lines. Flow cytometry reveals PTX abrogation of the radiation induced G2 arrest for both cell lines. PTX also prolongs G1 transit for both cell lines. Preliminary results are presented using a novel methylxanthine, lisofylline (LSF), which has similar cell cycle effects on G1 and G2 and achieves differential radiosensitization at micromolar concentrations that are sustainable in humans. CONCLUSION: This checkpoint-based strategy is a promising approach for achieving preferential radiosensitization of p53- tumors relative to p53+ normal tissues.

Cell Survival↗
PubMed1996

Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.

The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged prediction of alpha-helix and beta-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a beta beta alpha beta motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different alpha/beta secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure.

Amino Acid Sequence↗
PubMed1996

Surface conformational and linear epitopes on HPV-16 and HPV-18 L1 virus-like particles as defined by monoclonal antibodies.

A panel of 24 monoclonal antibodies (MAbs) was generated against human papillomavirus (HPV) types 16 and 18 L1 virus-like particles (VLPs). The MAbs were screened for reactivity to a variety of VLPs prepared from HPV-6, -11, -16, -18, -31, -33, -35, and -45, cottontail rabbit papillomavirus, bovine papillomavirus type 1, and a set of 35 overlapping 20-amino-acid peptides spanning the entire HPV-16 L1 gene. Type-specific linear and conformational surface epitopes were detected as well as several cross-reactive linear epitopes that showed various levels of cross-reactivity between different genital HPV and animal papillomavirus L1s. Most of the linear epitopes were mapped using synthetic peptides, and the epitopes were identified as being either surface or buried within the VLP as defined by the pattern of reactivity in ELISA using intact and disrupted VLP antigen. These MAbs may be useful reagents to help define neutralizing epitopes of HPV-16 and -18 when infectivity assays become available, and to define the regions of L1 that are exposed on the surface or buried within the assembled capsid.

Amino Acid Sequence↗