Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro.

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.

The activity of topoisomerase I is modulated by large T antigen during unwinding of the SV40 origin.

When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origin-containing DNA.

Topoisomerase I stimulates SV40 T antigen-mediated DNA replication and inhibits T antigen's ability to unwind DNA at nonorigin sites.

We have previously found that purified SV40 T antigen and topoisomerase I (topo I) bind to one another in vitro. In this report, we determined the effects of human topo I on T antigen-mediated DNA replication and investigated whether it altered T antigen's biochemical activities. Topo I stimulates DNA replication and especially increases the amounts of finished circular molecules. This protein had no effect on T antigen's ability to bind, distort, or unwind the origin of replication. However, unwinding of DNA by T antigen was strongly inhibited by topo I when it was initiated at sites other than the origin. We demonstrate that the presence of T antigen binding sites in DNA interfere with inhibition of unwinding by topo I. These results indicate that topo I may increase the specificity of unwinding by inhibiting the reaction at non-origin sites. Fragments of T antigen that bind to topo I abrogate topo I's inhibition of non-origin-dependent unwinding, indicating that topo I inhibits unwinding through a direct interaction with T antigen. We propose a model whereby T antigen and topo I function together at the origin to specifically unwind it and initiate DNA replication.

Hair analysis does not support hypothesized arsenic and chromium exposure from drinking water in Woburn, Massachusetts.

We hypothesized that residents of Woburn, Massachusetts, had been exposed to as much as 70 microg/l of arsenic (As) and 240 microg/l of chromium (Cr) in drinking water from municipal supply wells G and H. To test this hypothesis, we measured the concentrations of As and Cr in 82 hair samples donated by 56 Woburn residents. Thirty-six samples were cut between 1964 and 1979, the period during which wells G and H were in operation. The remainder were cut either before 1964 (1938-1963; n = 26) or after 1979 (1982-1994; n = 20). Washed hair samples were analyzed by instrumental neutron activation. Exposure to the well water--measured as access--was estimated using well pumping records and a model of the Woburn water distribution system. Our results show that access to wells G and H water was not significantly correlated (95% confidence interval) with As and Cr concentrations measured in the hair of Woburn residents, but As concentrations have declined significantly over the last half century. Linear regression of As concentrations (micrograms per gram) upon year of hair cut and access to wells G and H water yielded a standard coefficient for year of -0. 0074 +/- 0.0017 (standard error; p = 2.5 -multiple- 10(-5)) and -0.12 +/- 0.10 (p = 0.22) for access. The r2 value for the model was 0.19. The geometric mean concentrations (geometric standard deviation) of As and Cr in the hair of residents who had access (i.e., relative access estimate >0) to wells G and H water (n = 27) were 0.14 (2.6) and 2.29 (1.8) microg/g, respectively; the geometric mean concentrations of As and Cr in all of the hair samples from residents who did not have access (1938-1994; n = 55) were 0.13 (3.0) and 2.19 (2.0) microg/g, respectively.

Replication activity of JC virus large T antigen phosphorylation and zinc finger domain mutants.

The replication potential of the human polyomavirus JC virus (JCV) relative to that of the related monkey virus SV40 is limited, in part, by differences in the multifunctional T antigen (T Ag). Earlier genetic analyses of the SV40 T protein indicated that specific phosphorylation sites and a zinc finger motif are involved in the regulation of viral replication. The JCV and SV40 T Ags differ with respect to sequences encoding these functional domains, and in the present study mutational analysis of the JCV protein was conducted to assess the role that unconserved residues might play in the restricted lytic behavior of JCV. Amino acids Asn316 and His317 in the zinc finger domain and Thr664 and Glu666 in the carboxy-terminal phosphorylation domain were mutated to either an SV40-like residue or an alanine. Each of the mutant JCV genomes replicated with wild type efficiency suggesting that, unlike the case for SV40 T Ag, these amino acids are not critical to the regulation of viral replication. On the other hand, mutation of amino acid Thr125 within the amino-terminal phosphorylation domain abolished JCV DNA replication and viability. This site is conserved in the SV40 T Ag, and previous results have revealed that phosphorylation of this residue (Thr124) is required for T Ag replication function.

Identification of three new JC virus proteins generated by alternative splicing of the early viral mRNA.

The genome of the human polyomavirus JC Virus (JCV) encodes two regulatory proteins, large and small T antigen which are expressed early in a lytic infection, and three structural proteins, VP1, VP2, VP3, which are produced late in an infection. A fourth late protein, agnoprotein, may contribute to the assembly of the virion. In this study, we demonstrate the presence of three additional early proteins, T'135, T'136, and T'165, which are expressed in lytically-infected cells; T'135 is also readily detected in JCV transformants. The three species of T' are phosphoproteins generated via an alternative splicing mechanism. This mechanism involves the excision of a second intron from the large T mRNA using a common donor splice site at JCV nucleotide 4274 and three unique acceptor splice sites at nucleotides 2918, 2777 and 2704 for T'135, T'136 and T'165, respectively. The mutant JCV delta T' was created by converting the G at nucleotide 4274 to an A, thereby disrupting the consensus sequence of the shared donor splice site without altering the amino acid sequence of any early JCV protein. Upon transfection of permissive human brain cells, JCV delta T' replicated its DNA 10-fold less efficiently than did wild type JCV. Passage of extracts of the infected cells on to fresh human brain cells revealed that the expression of T antigen was greatly reduced and the presence of T' proteins was undetectable in the mutant versus wild type JCV-infected cells.

Converting the JCV T antigen Rb binding domain to that of SV40 does not alter JCV's limited transforming activity but does eliminate viral viability.

Two sets of mutations were introduced into a region of the JC virus (JCV) large tumor (T) antigen involved in binding the retinoblastoma susceptibility gene product (Rb). The first set converted the JCV sequences to those found in the corresponding region of the simian virus 40 (SV40) T antigen. The second set contained sequence changes predicted to abolish Rb binding. Each of these mutations was also inserted into a chimeric T antigen (MSTn) composed of JCV and SV40 sequences at its amino- and carboxy termini, respectively. The JCV T antigen is less efficient than its SV40 counterpart at transforming Rat2 cells and at binding Rb and viral DNA. These activities were altered in the two sets of mutants generated in this study. A JCV T antigen mutant having an SV40-like Rb-binding domain exhibited increased DNA binding activity while, unexpectedly, displaying decreased Rb binding and wild-type transforming behavior. A mutant T antigen that was unable to bind Rb exhibited decreased DNA binding and failed to transform Rat2 cells. Both mutants were defective for DNA replication and did not produce infectious virions. Additional phenotypic changes were observed when each mutation was introduced into the chimeric MSTn T antigen. As the oligomerization state of SV40 T antigen is known to influence several of its activities, including Rb binding, the quaternary structure of the T proteins used in this study was assessed by sucrose gradient sedimentation. The SV40 and chimeric MSTn T antigen sedimented as a mixture of monomers/dimers and higher oligomers, whereas the JCV T antigen sedimented predominantly as monomers/dimers; neither mutation in the T antigen Rb-binding motif affected the sedimentation profiles of the parental T proteins. Restricted biochemical activity of the JCV T protein relative to that of SV40 supports the suggestion that this regulatory protein contributes to the attenuation of the JCV lytic cycle.

Analysis of G418-selected Rat2 cells containing prototype, variant, mutant, and chimeric JC virus and SV40 genomes.

The human polyomavirus JC virus (JCV) is highly tumorigenic in rodents, but transforms cells in culture inefficiently. To explore the basis for JCV's restricted transforming behavior, nonpermissive Rat2 cells were contransfected with pSV2-neo (encodes G418 resistance) and viral DNAs including prototype, variant, and mutant JCV genomes and two JCV-SV40 chimeras. By selecting cells displaying G418 resistance, lines were established that contain viral DNA and exhibit a wide range of transformed phenotypes. The G418-resistant lines were tested for their ability to grow under anchorage-independent conditions, to overgrow a monolayer of untransformed cells, and to form dense colonies on plastic. Expression of the viral T and t proteins and interaction of T protein with the cellular anti-oncoprotein p53 were measured. Also determined was the number of intact viral early coding regions integrated within the cellular DNA. The results of these studies suggested that most of the G418-resistant lines failed to express JCV T protein above a minimum threshold level required for their conversion to a fully transformed phenotype. In anchorage-independent growth assays, higher levels of a 17-kDa T-related peptide in JCV transformants appeared to compensate for decreased T antigen levels. Comparison of the T to p53 ratios in the cell lysates suggested that the quaternary structure of the JCV protein differed from that of its SV40 counterpart in the T-p53 complex. The presence of multiple vs single integrated copies of the viral genome in the cells did not correlate with elevated T antigen expression or an enhanced transformation status.

Oral contraceptive use and histopathology of cancerous breasts in young women. Members of the U.K. National Case-Control Study Group.

A retrospective histopathological study of 300 women under 36 years of age was carried out to determine whether breast cancers occurring in oral contraceptive users showed any differences in pathological features compared with non-users. The patients belong to an age group in which an increased risk of cancer development has been reported following oral contraceptive usage. The incidence of non-neoplastic conditions in the residual breast was also studied in the two groups. There was little difference between breast cancers arising in pill users and non-users but in the residual non-neoplastic breast a decreased incidence of cysts and blunt duct adenosis was found in current users of the contraceptive pill. In contrast, lactational foci were found only in the breasts of pill users. The incidence of intraductal hyperplasia was not significantly different in the two groups.

Stress and cancer surveys: attitudes of participants in a case-control study.

STUDY OBJECTIVE: The aim was to ascertain whether personal interviews carried out for cancer case-control studies cause stress to participants. DESIGN: Retrospective postal questionnaires were sent to women at least three months after interview for a case-control study of the aetiology of cervical cancer. The questionnaire covered attitudes to taking part in the study, stress engendered by participation, whether any particular questions were distressing, factors relevant to the decision to participate, and the role of their doctor with respect to participation. SETTING: South East and South West Thames health regions, United Kingdom. PATIENTS: Patients were women aged 20-45 years at diagnosis with invasive cervical cancer, and population based controls. MEASUREMENTS AND MAIN RESULTS: The response rate was 90%. Nearly all respondents were glad they had participated, while only 2/226 regretted taking part. Half the respondents (115/226) perceived some actual benefit from taking part. The interview carried out in the case-control study was both long and detailed and included topics such as numbers of sexual partners and history of sexually transmitted diseases. As expected, the questions causing most concern to interviewees were those on number of sexual partners, but only 13% of participants were bothered by these questions and only 4% felt inclined to terminate the interview early. CONCLUSIONS: The lack of evidence of stress caused by this potentially difficult interview suggests that, in the hands of experienced interviewers, stress is unlikely to be caused by participation. Many participants felt that they had benefited from taking part. Doctors and ethics committees should find these results reassuring.

Benign villous adenomas of the ampulla of Vater.

One of the most unusual sites of isolated villous adenomas is the ampulla of Vater. Six patients with this neoplasm have recently been identified. The mean age was 68 years, and four were female. Four of the six were evaluated on several occasions before definitive diagnosis. Preoperative endoscopy correctly identified the lesions in two of three patients. Five patients underwent local resection and one a radical pancreaticoduodenectomy. Six benign neoplasms of the ampulla were found. In discussing this entity, of which there have been 38 reported cases in the literature, the role of local excision that can be combined with more complex procedures such as sphincteroplasty and duct implantation should be emphasized.

Evolving changes in the pathogenesis and treatment of the perforated gallbladder. A combined hospital study.

Perforation of the gallbladder occurred in 35 patients in this 6 year review, with a 2.3:1 male predominance in contrast with a female dominance in nonperforated acute cholecystitis. Thirty-three percent of patients with gallstones had a history of symptomatic cholelithiasis which emphasizes that if elective cholecystectomy had been performed, this complication could have been avoided. Further, a large number of cases (40 percent) were found to be of the acalculous variety which suggests a possible changing trend in the pathogenesis of perforated gallbladder. Cholecystectomy with intraoperative cholangiography and adequate drainage appears to be the procedure of choice, and aggressive operative intervention without delay is thought to contribute to the relatively low mortality of 8.6 percent in this series.

A randomized study of cholecystectomy with and without drainage.

Routine drainage of the bed of the gallbladder following cholecystectomy remains controversial. A prospectively randomized series of 100 instances of cholecystectomy for chronic and subsiding acute cholecystitis are reviewed. Postoperative fever was lowered, the need for dressing changes was virtually eliminated and hospital stay was shortened in patients who did not undergo drainage. No complications attributable to drain avoidance were seen.