Smoking, internalized heterosexism, and HIV disease management among male couples.

High rates of cigarette smoking have been observed among HIV-positive individuals. Smoking has been linked to HIV-related medical complications and non-AIDS defining cancers and negatively impacts on immune function and virologic control. Although internalized heterosexism has been related to smoking behaviors, little is known about associations between partners' reports of smoking, internalized heterosexism, and HIV medication management in male couples with HIV. A sample of 266 male couples completed baseline assessments for a cohort study examining relationship factors and HIV treatment. A computer-based survey assessed self-reported smoking behaviors, alcohol use, internalized heterosexism, and antiretroviral therapy (ART) adherence. HIV-positive men also provided blood samples to assess viral load. Approximately 30% of the sample reported that they are currently smoking cigarettes. After adjusting for demographic characteristics, men in a primary relationship with a partner who reported currently smoking had more than five-fold greater odds of reporting smoking. Higher levels of internalized heterosexism and financial hardship were each independently associated with greater odds of reporting smoking. Among HIV-positive men on ART (n = 371), having a partner who reported smoking was associated with almost three-fold greater odds of having a detectable viral load. Our findings add new support to the evidence of romantic partners influencing each other's health behaviors, and demonstrate an association between smoking and disease management within male couples. Future research should explore the interpersonal and social contexts of smoking in order to develop interventions that meet the unique needs of male couples.

Personal or relational? Examining sexual health in the context of HIV serodiscordant same-sex male couples.

Couples' ability to adopt a "we" orientation has been associated with optimal health outcomes. This study examined how personal and relational motivations are uniquely associated with unprotected anal intercourse (UAI), protected anal intercourse (PAI), and the absence of sexual activity within HIV-serodiscordant same-sex male couples. HIV-positive men and their HIV-negative partners (N = 116 couples, 232 men) completed questionnaires and HIV-positive men had blood drawn for viral load. Results of a multinomial logistic regression illustrated that sexual satisfaction was positively associated with PAI among HIV-negative partners and negatively associated with PAI among HIV-positive partners. Endorsing a "we" orientation was positively associated with PAI among HIV-positive partners. Findings suggest that HIV-positive partners who espouse a "we" orientation may be willing to forgo their personal interests to protect their HIV-negative partners from HIV transmission. Couples-based interventions are warranted to help strengthen relationship dynamics to enhance the sexual health of serodiscordant couples.

Cell transformation by the middle T-antigen of polyoma virus.

The polyoma virus region expressed early in the lytic cycle encodes three proteins, or T-antigens, that together cause the infected cell to enter the cell cycle and so provide a suitable cellular environment for replication of the viral genome. Under some circumstances infection does not kill the cell, but the T-antigens are still produced, resulting in the cell becoming transformed and tumorigenic. Most of this transforming action is exerted by the middle T-antigen, which has the ability to convert established cell lines to an oncogenic state. Middle T is a membrane bound polypeptide that interacts with a number of the proteins used by tyrosine kinase associated receptors to stimulate mitogenesis, so MT can be considered as a permanently active analogue of a receptor. Through a defined series of interactions, MT assembles a large multi-protein complex at the cell membrane, consisting of MT, the core dimer of protein phosphatase 2A, an src-family tyrosine kinase, and via phosphotyrosines, ShcA, phosphatidylinositol (3') kinase, and phospholipase Cgamma-1. Tyrosine phosphorylation stimulates PI3K and PLCgamma-1 enzymatic activity, and on ShcA creates binding sites for Grb2 with its associated Sos1 and Gab1. This activates p21(ras), and hence, the MAP kinase cascade. Consequently, MT can be used as a model for studying cell transformation and growth factor receptor signalling pathways.

ShcA and Grb2 mediate polyoma middle T antigen-induced endothelial transformation and Gab1 tyrosine phosphorylation.

Middle T antigen (PymT) is the principal transforming component of polyomavirus, and rapidly induces hemangiomas in neonatal mice. PymT, a membrane-associated scaffold, recruits and activates Src family tyrosine kinases, and, once tyrosine phosphorylated, binds proteins with PTB and SH2 domains such as ShcA, phosphatidylinositol 3-kinase (PI3K) and phospholipase Cgamma-1 (PLCgamma-1). To explore the pathways required for endothelial transformation in vivo, we introduced PymT mutant forms into mice. PymT variants unable to bind PI3K and PLCgamma-1 directly induced hemangiomas similarly to wild type, but a mutant unable to bind ShcA was transformation compromised. Requirement for a ShcA PTB domain- binding site was suppressed by replacing this motif in PymT with YXN sequences, which bind the Grb2 SH2 domain upon phosphorylation. Surprisingly, PymT recruitment of ShcA and Grb2 correlated with PI3K activation. PymT mimics activated receptor tyrosine kinases by forming a ShcA-Grb2-Gab1 complex, thus inducing Gab1 tyrosine phosphorylation, which itself is associated with PI3K. Therefore, PymT activation of ShcA-Grb2 signaling is critical for endothelial transformation, and PymT can stimulate Grb2 signaling to both the MAP kinase and PI3K pathways.

ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T-antigen.

ShcA and Grb2 are crucial components in signalling by most tyrosine kinase-associated receptors. How ever, it is not clear whether Grb2 bound directly to the receptor is equivalent to Grb2 associated via ShcA. We have used signalling stimulated by the middle T-antigen (MT) of polyoma virus to address this question. The two known Grb2-binding sites from murine ShcA, 313Y and 239/240YY, could functionally replace the MT ShcA-interacting region in transformation assays using Rat2 fibroblasts. This demonstrates that signal output from membrane-bound ShcA requires only these two sequences and the ShcA-binding site in MT does not recruit other signalling molecules. Two standard Grb2-interacting sequences, either from the EGF receptor or the ShcA 313Y region, could not replace the requirement for ShcA binding to MT, indicating an enhanced role for the ShcA 239/240YY motif. Sos1 and the docking protein Gab1 are brought into the MT complex through Grb2 association and this may be more effective using the 239/240YY sequence.

Minichromosome maintenance protein 2 expression in prostate: characterization and association with outcome after therapy for cancer.

The minichromosome maintenance (MCM) proteins are highly conserved proteins essential for initiating and regulating eukaryotic DNA replication. Recent studies have demonstrated the potential use of MCM proteins as markers of proliferation. We characterized the pattern of Mcm 2 staining in benign and malignant prostate tissues and examined the role of Mcm 2 expression in disease-free survival after surgery in men with localized prostate cancer. Tumors from 92 patients who underwent radical prostatectomy for prostate cancer (median follow-up of 54 months) were examined for Mcm 2 expression by immunohistochemistry using a monoclonal antibody. Prostate tissue from five men without histopathological evidence of prostate cancer was also stained for Mcm 2. Mcm 2 expression was quantified by calculating a labeling index, and patients were grouped according to degree of staining. An analysis of the association between Mcm 2 expression with traditional clinicopathological characteristics of prostate cancer was carried out. A Cox proportional hazards analysis was performed to determine whether Mcm 2 staining was a significant independent predictor of disease-free survival. Mcm 2 expression is low (<2%) and limited to the basal cell layer in nonmalignant prostate glands. Mcm 2 expression is consistently increased in malignant glands and is significantly associated with disease-free survival in univariate (P = 0.002) and multivariate (P = 0.01) analyses. Patients with high Mcm 2 expression exhibited shorter disease-free survival. Mcm 2 expression was not associated with any traditional clinical or pathological factors and therefore is an independent predictor of survival in these patients with prostate cancer. These data support evidence that Mcm 2 may serve as a novel proliferation marker in the prostate. Mcm 2 expression is an independent predictor of disease-free survival after definitive local therapy and has potential as a molecular marker for clinical outcome in prostate cancer.

Biologic factors and response to radiotherapy in carcinoma of the cervix.

Ionizing radiation has been used to treat cancers for a century. However, radioresistance remains a major problem in the clinic. Recent advances in the understanding of the molecular events that occur following ionizing radiation leading to DNA damage and repair, apoptosis, and cell cycle arrests suggest new ways in which the radiation response might be manipulated. Seventy-eight cases of carcinoma of the cervix of the same stage (II A and B) were analyzed retrospectively. All patients were treated with radiotherapy (RT) with a dose varying from 35 Gy to 50 Gy with 200 cGy per fraction. Subsequent to the completion of radiotherapy, all patients underwent surgery 4-6 weeks later. On histological examination of the surgical specimens, 51% of the cases (40) showed a complete response to therapy with no viable tumor cells. 49% of cases (38) had residual tumors ranging from a small focus to lesions extending through more than half the thickness of the cervical wall. p53 (mutant), bcl-2, p21 and bax proteins were studied on the paraffin sections of the biopsies (pretreatment) of those patients who failed to respond to RT and compared to similar studies on biopsies of patients who had a complete response to RT. In addition, the minichromosome maintenance (MCM) 2 proliferative marker was also done on all cases. Expression of all proteins was done using immunohistochemsitry. In the radioresistant cases, 15% (six cases) showed positivity for bcl-2 and p21, respectively, and 34% (13 cases) showed mutant p53. None of the radiosensitive tumors were positive for the above proteins. 75% of the radiosensitive tumors (30 cases) were positive for the bax antibody, whereas 81% of the radioresistant tumors (31 cases) were negative for bax. The MCM2 proliferative marker was positive in > 80% of cells in 81.5% of radioresistant tumors (31 cases) as compared to < 40% of cells that were positive in 70% of radiosensitive tumors (28 cases). The P-value for the biological markers was calculated using the chi-squared test, and was highly significant (P < 0.01) for all the parameters tested. However, there was no statistical significance by univariate analysis when the dose of radiation was analyzed with respect to the markers and the histological response. There was also no correlation between the radiation response and timing of surgery. The above data strongly suggest that bax, along with proliferative markers, could play a role in determining which tumors are likely to respond to radiation therapy. The presence of bcl-2, p21 and p53 could also be related to radioresistance of the tumors.

Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation.

The Raf-1 kinase plays a key role in relaying proliferation signals elicited by mitogens or oncogenes. Raf-1 is regulated by complex and incompletely understood mechanisms including phosphorylation. A number of studies have indicated that phosphorylation of serines 259 and 621 can inhibit the Raf-1 kinase. We show that both serines are hypophosphorylated during early mitogenic stimulation and that hypophosphorylation correlates with peak Raf-1 activation. Concentrations of okadaic acid that selectively inhibit protein phosphatase 2A (PP2A) induce phosphorylation of these residues and prevent maximal activation of the Raf-1 kinase. This effect is mediated via phosphorylation of serine 259. The PP2A core heterodimer forms complexes with Raf-1 in vivo and in vitro. These data identify PP2A as a positive regulator of Raf-1 activation and are the first indication that PP2A may support the activation of an associated kinase.

Association between src-kinases and the polyoma virus oncogene middle T-antigen requires PP2A and a specific sequence motif.

Polymoma virus encodes a potent oncogene, the middle T-antigen (MT), that induces cell transformation by copying the actions of tyrosine kinase associated growth factor receptors. A crucial component of MT transformation is its ability to bind and stimulate the activity of src-family kinases. However, the mechanism by which this is achieved remains unclear. Tyrosine phosphorylation of MT by src-kinases then provides binding sites for SH2 and PTB domain containing molecules in a paradigm of receptor action. We present evidence here that the MT/src complex contains equi-molar amounts of PP2A, and that phosphatase activity may be required for the interaction of MT with both PP2A and the src-family. PP2A, then, is a necessary component of the MT-src complex. We also show that two motifs in the 185 to 210 region of MT, each consisting of a basic area followed by a serine or threonine, are essential for interaction with src-kinases, but not PP2A. The spacing between the serine or threonine and the basic sequence also appears to be important. Substituting a cysteine residue in place of Thr203 in MT has no affect on the binding of pp60c-src, showing that these sites interact with src-kinases by a novel mechanism that does not require phosphorylation.

Polyomavirus middle T antigen as a probe for T cell antigen receptor-coupled signaling pathways.

Stimulation of the T cell antigen receptor (TCR) triggers a complex series of signaling events that culminate in T cell activation and proliferation. The complex structure of the TCR has hindered efforts to link specific signaling events induced by TCR cross-linkage to downstream activation responses, such as interleukin-2 (IL-2) gene transcription. Previous studies have shown that the polyomavirus-derived oncoprotein, middle T antigen (mT), transforms rodent fibroblasts by interacting with and activating several cytoplasmic signaling proteins (Src kinases, phospholipase C (PLC)-gamma1, Shc, and phosphoinositide 3-kinase (PI3-K) implicated in cell growth control. In this study, we demonstrate that expression of mT activates Jurkat T cells, as measured by increases in IL-2 promoter- and NFAT (nuclear factor of activated T cells)-dependent reporter gene transcription. The transcriptional response provoked by mT was blocked by the immunosuppressive drug FK506, a potent inhibitor of TCR-mediated IL-2 gene expression. Mutations that disrupted the binding of mT to Src kinases or PLC-gamma1 abrogated the ability of mT to deliver the signals needed for IL-2 promoter activation. In contrast, a mT mutant that failed to bind PI3-K induced a markedly elevated transcriptional response in Jurkat cells, whereas mutation of the Shc binding site in mT had little effect on the transactivating potential of this viral oncoprotein. Additional studies demonstrated that the association of mT with PLC-gamma1 was necessary and sufficient to activate both Ca2+- and Ras-dependent signaling cascades in Jurkat cells. These results indicate that PLC-gamma1 activation plays pivotal and pleiotropic roles in the stimulation of IL-2 gene expression, whereas activation of PI3-K negatively modulates this response in Jurkat T cells.

Multimerization of polyomavirus middle-T antigen.

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.

The role of the Shc phosphotyrosine interaction/phosphotyrosine binding domain and tyrosine phosphorylation sites in polyoma middle T antigen-mediated cell transformation.

The phosphotyrosine interaction (PI)/phosphotyrosine binding (PTB) domain of Shc binds specific tyrosine-phosphorylated motifs found on activated growth factor receptors and proteins such as polyoma virus middle T antigen (MT). Phenylalanine 198 (Phe198) has been identified as a crucial residue involved in the interaction of the Shc PI/PTB with phosphopeptides. In NIH 3T3 cells expressing MT, p52 Shc carrying the F198V mutation is weakly phosphorylated and does not bind MT or Grb2. Overexpression of the PI/PTB domain alone as Shc amino acids 1-238 acted in a dominant interfering fashion blocking MT-induced transformation. However, expression of a slightly longer construct, Shc 1-260, which encompasses Tyr239/Tyr240, a novel Shc tyrosine phosphorylation site, did not block transformation. This was found to be due to the ability of Shc 1-260 to become tyrosine-phosphorylated and bind Grb2. Furthermore, full-length Shc in which Tyr239/Tyr240 had been mutated to phenylalanine did not become tyrosine-phosphorylated or bind Grb2 but did inhibit colony formation in soft agar. Conversely, p52 Shc carrying a mutation in the other tyrosine phosphorylation site, Tyr317, became heavily tyrosine-phosphorylated, bound Grb2, and gave rise to colonies in soft agar.

pp60c-src binding to polyomavirus middle T-antigen (MT) requires residues 185 to 210 of the MT sequence.

Interaction with the src family of tyrosine kinases is crucial to the transforming action of polyomavirus middle T-antigen (MT). Association with MT activates the tyrosine kinase activity of pp60(c-src) and, through subsequent MT phosphorylation, creates binding sites for signalling molecules whose stimulation culminates in cell transformation. Despite this importance, and many studies, little is known of the mechanisms by which pp60(c-src) binds to MT. We report here isolation of the first MT mutants that disrupt pp60(c-src) binding without affecting the interaction between MT and protein phosphatase 2A (PP2A). Through deletion analysis we established that interaction with pp60(c-src) requires the sequences between amino acids 185 and 210 of MT, but these residues have no effect on PP2A binding. Cells expressing these mutants showed few altered properties, indicating that the PP2A-MT interaction alone has little influence on cell phenotype. Subcellular location of these mutant MT molecules was indistinguishable by immunofluorescence analysis from that of wild-type MT but was altered markedly on loss of PP2A binding. This suggests a possible role for PP2A in specifying subcellular distribution.

Polyomavirus middle-T antigen lacking a membrane anchor sequence accumulates in the nucleus.

Three proteins expressed early in the replicative cycle of polyomavirus also play an essential role during virus-mediated tumorigenesis. One of the proteins, middle-T antigen, has been shown to bind cellular proteins involved in cell signalling such as c-Src, phosphatase 2A, phosphatidylinositol 3-kinase and SHC. Association of middle-T antigen with cellular membranes has been shown to be essential for middle-T-mediated cell transformation. A mutant virus encoding a truncated form of middle-T lacking a carboxy-terminal hydrophobic sequence mediating membrane association is not oncogenic. This mutant middle-T still binds phosphatase 2A through amino-terminal sequences common to small-and middle-T and is localized in the nucleus, although the protein does not contain a classical nuclear targeting sequence. Mutations introduced into the amino-terminal domain affecting the ability of truncated middle-T to bind phosphatase 2A prevented accumulation of the protein in the nucleus and led to localization in the cytoplasm. This suggests that nuclear localization of truncated middle-T may be a consequence of binding to phosphatase 2A.

Polyoma virus middle T antigen: meddler or mimic?

Polyoma virus middle T antigen duplicates the actions of growth-factor receptors in binding the signalling molecules phosphatidylinositol 3'-OH kinase and Shc. These properties indicate that middle T is mitogenic and may be required to overcome inhibition of DNA replication during the lytic life cycle of the virus.

Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc.

Polyoma virus middle T-antigen converts normal fibroblasts to a fully transformed, tumorigenic phenotype. It achieves this, at least in part, by binding and activating one of the non-receptor tyrosine kinases, pp60c-src, pp62c-yes or pp59c-fyn (reviewed in refs 2 and 3). As a result, middle T-antigen itself is phosphorylated on tyrosine residues, one of which (Tyr 315) acts as a binding site for the SH2 domains of phosphatidylinositol-3'OH kinase 85K subunit. Here we show that another tyrosine phosphorylation site in middle T-antigen (Tyr 250; refs 4, 5) acts as a binding region for the SH2 domain of the transforming protein Shc. This results in Shc also becoming tyrosine-phosphorylated and binding to the SH2 domain of Grb2 (ref. 10). This probably stimulates p21ras activity through the mammalian homologue of the Drosophila guanine-nucleotide-exchange factor Sos (reviewed in ref. 11). We suggest that middle T-antigen transforms cells by acting as a functional homologue of an activated tyrosine kinase-associated growth-factor receptor.

Novel monoclonal antibodies that differentiate between the binding of pp60c-src or protein phosphatase 2A by polyomavirus middle T antigen.

Fourteen pGEX plasmids that express defined regions of polyomavirus middle T antigen in bacteria have been constructed. These polypeptides have been used to generate 18 new monoclonal antibodies directed against the unique portion of middle T and to map the approximate position of the antibody recognition sites onto the protein sequence. All of the antibodies effectively immunoprecipitate middle T and the associated 60- and 35-kDa components of protein phosphatase 2A. Four of the antibodies, however, do not react with middle T when it is bound to pp60c-src. These four probably bind to amino acids 203 to 218 of the middle T protein sequence, which are encoded by the mRNA immediately 3' to the splice junction that creates the C-terminal unique region. This suggests that additional middle T sequences are required for middle T's interaction with pp60c-src than are needed for its binding to protein phosphatase 2A. The antibodies localize this extra region and provide a means of distinguishing between these two associations.

Cooperation of structural proteins during late events in the life cycle of polyomavirus.

The polyomavirus minor late capsid antigen, VP2, is myristylated on its N-terminal glycine, this modification being required for efficient infection of mouse cells. To study further the functions of this antigen, as well as those of the other minor late antigen, VP3, recombinant baculoviruses carrying genes for VP1, VP2, and VP3 have been constructed and the corresponding proteins have been synthesized in insect cells. A monoclonal antibody recognizing VP1, alpha-PyVP1-A, and two monoclonal antibodies against the common region of VP2 and VP3, alpha-PyVP2/3-A and alpha-PyVP2/3-B, have been generated. Reactions of antibodies with antigens were characterized by indirect immunofluorescence, immunoprecipitation, and immunoblot analysis. Immunofluorescent staining of mouse cells infected with polyomavirus showed all antigens to be localized in nuclei. When the late polyomavirus proteins were expressed separately in insect cells, however, only VP1 was efficiently transported into the nucleus; VP2 was localized discretely around the outside of the nucleus, and VP3 exhibited a diffused staining pattern in the cytoplasm. Coexpression of VP2, or VP3, with VP1 restored nuclear localization. Immunoprecipitation of infected mouse cells with either anti-VP1 or anti-VP2/3 antibodies precipitated complexes containing all three species, consistent with the notion that VP1 is necessary for efficient transport of VP2 and VP3 into the nucleus. Purified empty capsid-like particles, formed in nuclei of insect cells coinfected with all three baculoviruses, contained VP2 and VP3 proteins in amounts comparable to those found in empty capsids purified from mouse cells infected with wild-type polyomavirus. Two-dimensional gel analysis of VP1 species revealed that coexpression with VP2 affects posttranslational modification of VP1.

The role of nucleoplasmin in chromatin assembly and disassembly.

Nucleoplasmin is the most abundant nuclear protein in Xenopus oocytes and eggs. The term 'molecular chaperone' was coined to describe its role in the assembly of the nucleosome subunits of chromatin. Although histones and DNA can self-assemble into nucleosomes, nucleoplasmin can facilitate this process in vitro by competing against non-specific charge interactions. In vivo nucleoplasmin binds histones H2A and H2B and transfers them to DNA. Another acidic nuclear protein, N1, binds and transfers histones H3 and H4. Nucleoplasmin has at least one other role in modulating chromatin structure in Xenopus eggs. It is required for the first stage of sperm chromatin decondensation. It binds and removes sperm basic proteins and replaces them by histones H2A and H2B, again forming nucleosomes, and resulting in decondensation of the compacted sperm chromatin. In addition we propose that the properties of the nuclear localization signal of nucleoplasmin can be explained by a model in which heat shock cognate protein hsc70 has a chaperone role in signal presentation during nuclear transport.