A case-control study of hepatitis C virus and its interaction with hepatitis B virus on the development of hepatocellular carcinoma in the USA

Objective. The aim of this study was to assess the independent risk of hepatitis C virus (HCV) infection in the development of hepatocellular carcinoma (HCC). The independent risk of hepatitis B virus (HBV), its interaction with hepatitis C virus and the association with other risk factors were examined.^ Methods. A hospital-based case-control study was conducted between January 1994 and December 1995. We enrolled 115 pathologically confirmed HCC patients and 230 nonliver cancer controls, who were matched by age ($\pm$5 years), gender, and year of diagnosis. Both cases and controls were recruited from The University of Texas M. D. Anderson Cancer Center at Houston. The risk factors were collected through personal interviews and blood samples were tested for HCV and HBV markers. Univariate and multivariate analyses were performed through conditional logistic regression.^ The prevalence of anti-HCV positive is 25.2% in HCC cases compared to 3.0% in controls. The univariate analysis showed that anti-HCV, HBsAg, alcohol drinking and cigarette smoking were significantly associated with HCC, however, family history of cancer, occupational chemical exposure, and use of oral contraceptive were not. Multivariate analysis revealed a matched odds ratio (OR) of 10.1 (95% CI 3.7-27.4) for anti-HCV, and an OR of 11.9 (95% CI 2.5-57.5) for HBsAg. However, dual infection of HCV and HBV had only a thirteen times increase in the risk of HCC, OR = 13.9 (95% CI 1.3-150.6). The estimated population attributable risk percent was 23.4% for HCV, 12.6% for HBV, and 5.3% for both viruses. Ever alcohol drinkers was positively associated with HCC, especially among daily drinkers, matched OR was 5.7 (95% CI 2.1-15.6). However, there was no significant increase in the risk of HCC among smokers as compared to nonsmokers. The mean age of HCC patients was significantly younger among the HBV(+) group and among the HCV(+)/HBV(+) group, when compared to the group of HCC patients with no viral markers. The association between past histories of blood transfusion, acupuncture, tattoo and IVDU was highly significant among the HCV(+) group and the HBV(+)/HCV(+) group, as compared to HCC patients with no viral markers. Forty percent of the HCC patients were pathologically or clinically diagnosed with liver cirrhosis. Anti-HCV(+) (OR = 3.6 95% CI 1.5-8.9) and alcohol drinking (OR = 2.7 95% CI 1.1-6.7), but not HBsAg, are the major risk factors for liver cirrhosis in HCC patients.^ Conclusion. Both hepatitis B virus and hepatitis C virus were independent risk factors for HCC. There was not enough evidence to determine the interaction between both viruses. Only daily alcoholic drinkers showed increasing risk for HCC development, as compared to nondrinkers. ^

Rescue from apoptosis in early (CD34 selected) versus late (CD34 unselected) human hematopoietic cells by VLA-4 and VCAM-1 dependent adhesion to bone marrow stromal cells

The interaction of hematopoietic precursor cell with bone marrow stromal cells is assumed to be important to the survival of hematopoietic precursor cells during hematopoietic cell long-term culture. Early hematopoietic stem cells are preferentially found within the stromal adherent cell fraction in primary long-term bone marrow cultures. The purpose of this dissertation was to understand the molecular mechanisms that govern these interactions for the regulation of survival and proliferation of early versus late hematopoietic cells.^ Monoclonal antibodies to the VLA-4 recognize the alpha4 beta1 integrin receptor on human hematopoietic cells. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34 selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34 selected cells was shown to induce apoptosis of CD34 selected cells in these CD34 selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34 selected. Since there is no difference between the alpha4 beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+ selected precursor cells proliferate at a higher rate when these cells are plated on recombinant VCAM-1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function which results from the anchorage-dependent growth of the CD34 selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage-independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy. ^

Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

Cell surface $\beta$-1,4-galactosyltransferase function during invasion and metastasis

Metastasis is the complex process of tumor cell spread which is responsible for the majority of cancer-related deaths. Metastasis necessitates complex phenotypic changes, many of which are mediated by changes in the activities of cell surface molecules. One of these is cell surface $\beta$1,4-galactosyltransferase (GalTase), which is elevated on more highly metastatic cells. In this study, both molecular and biochemical methods were used to perturb and manipulate cell surface GalTase levels on K1735 murine melanoma cell lines in order to examine its function in metastasis.^ As expected, highly metastatic K1735 variants have higher cell surface GalTase than poorly metastatic variants. Stably transfected K1735 cell lines that overexpress surface GalTase were created. These cell lines were assayed for metastatic ability using an invasion chamber with Matrigel-coated filter inserts. Cells with increased surface GalTase were uniformly more invasive than neo transfected controls. With multiple cell lines, there was a direct correlation (r = 0.918) between surface GalTase activity and invasiveness. Homologous recombination was used to create K1735 cells with decreased levels of surface GalTase. These cells were uniformly less invasive than controls. Cell surface GalTase was inhibited using two different biochemical strategies. In both cases, inhibition of surface GalTase led to a decrease in in vivo metastatic ability of K1735 cells. This is the first direct experimental evidence addressing GalTase function in metastasis. These data provide several lines of independent evidence which show that cell surface GalTase facilitates invasion and metastasis. ^

Development of a rapid bioassessment method and index of biotic integrity for estuaries located along the northwest Gulf of Mexico

Recently it has been proposed that the evaluation of effects of pollutants on aquatic organisms can provide an early warning system of potential environmental and human health risks (NRC 1991). Unfortunately there are few methods available to aquatic biologists to conduct assessments of the effects of pollutants on aquatic animal community health. The primary goal of this research was to develop and evaluate the feasibility of such a method. Specifically, the primary objective of this study was to develop a prototype rapid bioassessment technique similar to the Index of Biotic Integrity (IBI) for the upper Texas and Northwestern Gulf of Mexico coastal tributaries. The IBI consists of a series of "metrics" which describes specific attributes of the aquatic community. Each of these metrics are given a score which is then subtotaled to derive a total assessment of the "health" of the aquatic community. This IBI procedure may provide an additional assessment tool for professionals in water quality management.^ The experimental design consisted primarily of compiling previously collected data from monitoring conducted by the Texas Natural Resource Conservation Commission (TNRCC) at five bayous classified according to potential for anthropogenic impact and salinity regime. Standardized hydrological, chemical, and biological monitoring had been conducted in each of these watersheds. The identification and evaluation of candidate metrics for inclusion in the estuarine IBI was conducted through the use of correlation analysis, cluster analysis, stepwise and normal discriminant analysis, and evaluation of cumulative distribution frequencies. Scores of each included metric were determined based on exceedances of specific percentiles. Individual scores were summed and a total IBI score and rank for the community computed.^ Results of these analyses yielded the proposed metrics and rankings listed in this report. Based on the results of this study, incorporation of an estuarine IBI method as a water quality assessment tool is warranted. Adopted metrics were correlated to seasonal trends and less so to salinity gradients observed during the study (0-25 ppt). Further refinement of this method is needed using a larger more inclusive data set which includes additional habitat types, salinity ranges, and temporal variation. ^

Targeting of {\it HER\/}-{\it 2\/}/{\it neu\/} with E1A gene therapy

Amplification or overexpression of HER-2/neu has been demonstrated in human cancers of the ovary, breast, lung and correlated with chemoresistance and poor clinic prognosis. We have previously found that the adenovirus type 5 early region 1A (E1A) gene product can repress the overexpression and suppress the tumorigenic potential of HER-2/neu-overexpressing cancer cells. In addition, E1A has been reported to induce apoptosis and inhibit the metastatic potential of tumor cells. Therefore, E1A could be considered as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. To develop an efficient HER-2/neu-targeting gene therapy with E1A, adenoviral vector or cationic liposome was used to introduce E1A into human ovarian, breast and lung cancer cells. Successful therapeutic effects were achieved.^ A replication-deficient adenovirus containing the E1A gene, Ad.E1A(+), was used to infect HER-2/neu-overexpressing human ovarian cancer cell line. Ovarian cancer growth in vitro and colony formation in soft agarose were greatly inhibited.^ To examine tumor suppressor function of E1A in breast cancer, we introduced E1A in vitro by adenovirus into both HER-2/neu-overexpressing and low-expressing human breast cancer cell lines. In HER-2/neu-overexpressing cells, E1A greatly inhibited tumor cell growth in vitro and colony formation in soft agarose. However, in low HER-2/neu expressing cancer cell lines, E1A could only reduce colony formation in soft agarose but had no significant effect on cell growth in monolayer, indicating different effects of E1A in these two types of cancer cells. To test the local therapeutic efficacy of E1A, we used either adenovirus- or liposome-mediated E1A gene delivery systems in an orthotopic breast cancer animal model.^ To test the therapeutic efficacy of systemically-delivered E1A in vivo lung cancer, we treated mice bearing intratracheal lung cancer by i.v. tail injections of Ad.E1A(+). As a result, Ad.E1A(+) suppressed HER-2/neu overexpression and inhibited intratracheal lung cancer growth. However, no significant tumor suppression effect of Ad.E1A(+) was observed in mice bearing HER-2/neu low expressing cell line when the same therapeutic procedure was followed. (Abstract shortened by UMI.) ^

Osteopontin (OPN) and neoplastic disease: Circulating levels and tissue distribution

OPN is a secreted phosphate containing protein which is expressed by osteoblasts and a variety of other cells in vivo. Data from in vitro studies has accumulated which relates OPN to cellular transformation. We hypothesize that OPN expression is associated with neoplastic disease in humans as suggested by cell culture models. The overall objective of the current study was to determine the tissue distribution of OPN in human malignancy and to determine whether or not a correlation exists between OPN serum levels and malignancy. At the inception of this project, no study had been made demonstrating the relevance of OPN expression with naturally occurring neoplastic disease in humans. To date, few studies have reported OPN distribution in human neoplasia and are limited by either the number of specimens analyzed or the technique used in analysis. In this dissertation study, OPN was purified from human milk and $\alpha$-OPN antiserum developed and characterized. Following antibody development, the distribution and prevalence of OPN in human oral squamous cell carcinoma and human prostate carcinoma was evaluated using immunohistochemical localization. OPN immunolocalization was found in a high percentage of oral epithelial dysplasia and oral squamous cell carcinoma in humans. One oral squamous cell carcinoma cells line, UMSCC-1, was found to express OPN mRNA using Northern blotting. OPN localized to a high percentage of primary prostate adenocarcinomas. OPN localized to 52% of androgen dependent cases and 100% of androgen independent cases. Androgen dependent cell lines such as LNCap and NbE showed minimal OPN mRNA expression while the androgen independent lines C4-2 and PC3 produced ample OPN mRNA. An OPN sandwich assay was developed and used to determine the serum level of OPN in normal males, patients with BPH (benign prostate hypertrophy), and patients with prostate carcinoma. No statistically significant difference was found in OPN serum levels among the three groups. However, a trend of increasing OPN in the serum was noted in patients with BPH and prostate cancer. ^

drp, A novel cell density regulated protein

Growing cells are continuously processing signals of all varieties and responding to these signals by changes in cellular gene expression. One signal that cells in close proximity relay to each other is cell-cell contact. Non-transformed cells respond to cell-cell contact by arrest of growth and entry into G$\sb0,$ a process known as contact inhibition. Transformed cells do not respond to contact inhibition and continue to grow to high cell density, forming foci when in cell culture and tumors in the living organism. The events surrounding the generation, transduction, and response to cellular contact are poorly understood. In the present study, a novel gene product, drp, is shown to be expressed at high levels in cultured cells at high cell density. This density regulated protein, drp, has an apparent molecular weight of 70 kDa. Northern analysis shows drp to be highly expressed in cardiac and skeletal muscle and least abundant in lung and kidney tissues. By homology to two independently derived sequence tagged sites (STSs) used in the human genome project, drp or a closely related sequence maps to human chromosome 12. Density-dependent increases in drp expression have been demonstrated in six different cell lines including NIH 3T3, Hela and a human teratocarcinoma cell line, PA-1. Cells exhibit increased drp expression both when they are plated at increasing concentrations per unit area, or plated at low density and allowed to grow naturally to higher cell density. Cells at high density can exhibit several phenotypes including growth arrest, accumulation of soluble factors in the media, and increased numbers of cell contacts. Growth arrest by serum starvation or TGF-$\beta$ treatment fails to produce an increase in drp expression. Similarly, treatment of low density cells with conditioned media from high density cells fails to elicit drp expression. These results argue that neither soluble factors accumulated or expressed at high density nor simple exit from the cell cycle is sufficient to produce an increase in drp expression. The expression of drp appears to be uniquely regulated by cell density alone. ^

Tumors in the contralateral breast following radiation therapy for breast cancer

The proportional distribution of independent malignant tumors in the contralateral breast following treatment for breast cancer was investigated to assess the influence of scattered radiation as a cause of these tumors. In a population of 172 patients the proportion of contralateral tumors in each quadrant and the center (the nipple-areolar complex) was compared with the expected, or natural, distribution found in the general population, in the absence of radiation. The observed/expected ratio for contralateral tumors was 1.43 for the upper-inner quadrant; 0.97, lower-inner quadrant; 1.51, center; 0.76, upper-outer quadrant; and 0.64, lower-outer quadrant. In each quadrant, except the lower-inner, the observed/expected ratio differed from 1.00 with statistical significance at the 5% level (one-tail). The same analysis, stratified by age and menopausal status, showed a similar shift of tumors, with more than expected in the inner quadrants and center and less than expected in the outer quadrants, although the results did not show statistical significance at the 5% level for all strata. For each patient the mean absorbed radiation dose for each quadrant and center of the breast was estimated, based on measurements in a tissue-equivalent phantom. Among patients the doses ranged from 0.5 to 8 Gy; within individuals, doses to the inner quadrants typically were a factor of three times higher than doses to the outer quadrants. The results suggest that radiation may be a risk factor for contralateral breast tumors and warrants further investigation. ^

Identification of the signal pathways modulated by expression of p210$\rm\sp{bcr-abl}$ and the inhibition of p210$\rm\sp{bcr-abl}$ transforming activity by polypeptides interacting with p210$\rm\sp{bcr-abl}$ in murine myeloid 32D cells

The Bcr-Abl fusion oncogene which resulted from a balanced reciprocal translocation between chromosome 9 and 22, t(9;22)(q11, q34), encodes a 210 KD elevated tyrosine specific protein kinase that is found in more than 95 percent of chronic myelogenous leukemia patients (CML). Increase of level of phosphorylation of tyrosine is observed on cell cycle regulatory proteins in cells overexpressing the Bcr-Abl oncogene, which activates multiple signaling pathways. In addition, distinct signals are required for transforming susceptible fibroblast and hematopoietic cells, and the minimal signals essential for transforming hematopoietic cells are yet to be defined. In the present study, we first established a tetracycline repressible p210$\rm\sp{bcr-abl}$ expression system in a murine myeloid cell line 32D c13, which depends on IL3 to grow in the presence of tetracycline and proliferate independent of IL3 in the absence of tetracycline. Interestingly, one of these sublines does not form tumors in athymic nude mice suggesting that these cells may not be completely transformed. These cells also exhibit a dose-dependent growth and expression of p210$\rm\sp{bcr-abl}$ at varying concentrations of tetracycline in the culture. However, p210$\rm\sp{bcr-abl}$ rescues IL3 deprivation induced apoptosis in a non-dose dependent fashion. DNA genotoxic damage induced by gamma-irradiation activates c-Abl tyrosine kinase, the cellular homologue of p210$\rm\sp{bcr-abl},$ and leads to activation of p38 MAP kinase in the cells. However, in the presence of p210$\rm\sp{bcr-abl}$ the irradiation failed to activate the p38 MAP kinase as examined by an antibody against phosphorylated p38 MAP kinase. Similarly, an altered tyrosine phosphorylation of the JAK1-STAT1 pathways was identified in cells constitutively overexpressing p210$\rm\sp{bcr-abl}.$ This may provided a molecular mechanism for altered therapeutic response of CML patients to IFN-$\alpha.$^ Bcr-Abl oncoprotein has multiple functional domains which have been identified by the work of others. The Bcr tetramerization domain, which may function to stabilize the association of the Bcr-Abl with actin filaments in p210$\rm\sp{bcr-abl}$ susceptible cells, are essential for transforming both fibroblast and hematopoietic cells. We designed a transcription unit encoding first 160 amino acids polypeptide of Bcr protein to test if this polypeptide can inhibit the transforming activity of the p210$\rm\sp{bcr-abl}$ oncoprotein in the 32D c13 cells. When this vector was transfected transiently along with the p210$\rm\sp{bcr-abl}$ expression vector, it can block the transforming activity of p210$\rm\sp{bcr-abl}.$ On the other hand, the retinoblastoma tumor suppressor protein (Rb), a naturally occurring negative regulator of the c-Abl kinase, the cellular homologue of Bcr-Abl oncoprotein, binds to and inhibits the c-Abl kinase in a cell cycle dependent manner. A polypeptide obtained from the carboxyl terminal end of the retinoblastoma tumor suppressor protein, in which the nuclear localization signal was mutated, was used to inhibit the kinase activity of the p210$\rm\sp{bcr-abl}$ in the cytoplasm. This polypeptide, called Rb MC-box, and its wild type form, Rb C-box, when overexpressed in the 32D cells are mainly localized in the cytoplasm. Cotransfection of a plasmid transcription unit coding for this polypeptide and the gene for the p210$\rm\sp{bcr-abl}$ resulted in reduced plating efficiency of p210$\rm\sp{bcr-abl}$ transfected IL3 independent 32D cells. Together, these results may lead to a molecular approach to therapy of CML and an in vitro assay system to identify new targets to which an inhibitory polypeptide transcription unit may be directed. ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

Molecular analysis of the human $\gamma$-chain T cell receptor genes

In our studies we have focused on the issue of variability and diversity of the $\gamma$ (or $\delta)$ chain T cell receptor (TCR) genes by studying cDNA transcripts in peripheral blood mononuclear cells or $\gamma\delta$ TCR+ T cell clones. The significance of these studies lies in the better understanding of the molecular biology of the $\gamma\delta$ T cell receptor as well as in answering the question whether certain molecular forms predominate in $\gamma\delta$ T cells exhibiting specific immunologic functions. We establish that certain $\gamma$-chain TCR genes exhibit particular patterns of rearrangements in cDNA transcripts in normal individuals. V$\gamma$I subgroup were shown to preferentially rearrange to J$\gamma$2C$\gamma$2 gene segments. These preferential VJC rearrangements, may have implications regarding the potential for diversity and polymorphism of the $\gamma$-chain TCR gene. In addition, the preferential association of V$\gamma$I genes with J$\gamma$2C$\gamma$2, which encode a non-disulfide-linked $\gamma\delta$ TCR, suggests that $\gamma$ chains utilizing V$\gamma$I are predominantly expressed as non-disulfide-linked $\gamma\delta$ TCR heterodimers. The implications of this type of expression remain to be determined. We identified two alternative splicing events of the $\gamma$-chain TCR genes occurring in high frequency in all the normal individuals examined. These events may suggest additional mechanisms of regulation and control as well as diversification of $\gamma\delta$ TCR gene expression. The question whether particular forms of $\gamma$ or $\delta$-chain TCR genes are involved in HLA Class I recognition by specific $\gamma\delta$ cytotoxic T cell clones was addressed. Our results indicated that the T cell clones expressed identical $\gamma$ but distinct $\delta$-chains suggesting that the specificity for recognition of HLA-A2 or HLA-A3 may be conferred by the $\delta$-chain TCR. The issue of the degree of diversity and polymorphism of the $\delta$-chain TCR genes in a patient with a primary immunodeficiency (Omenn's syndrome) was addressed. A limited pattern of rearrangements in peripheral blood transcripts was found, suggesting that a limited $\gamma\delta$ TCR repertoire may be expressed in this particular primary immunodeficiency syndrome. Overall, our findings suggest that $\delta$-chain TCR genes exhibit the potential for significant diversity and that there are certain preferential patterns of expression that may be associated with particular immunologic functions. ^

Design, synthesis and biological evaluation of 5$\sp\prime$-mononucleotide prodrugs: Strategies to introduce nucleotides into cells

The neutral bis ((pivaloyloxy)methyl) (PIV$\sb2\rbrack$ derivatives of FdUMP, ddUMP, and AZTMP were synthesized as potential membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. These compounds were designed to enter cells by passive diffusion and revert to the parent nucleotides after removal of the PIV groups by hydrolytic enzymes. These prodrugs were prepared by condensation of FUdR, ddU, and AZT with PIV$\sb2$ phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP were stable in the pH range 1.0-4.0 (t$\sb{1/2} = {>}$100 h). They were also fairly stable at pH 7.4 (t$\sb{1/2} = {>}$40 h). In 0.05 M NaOH solution, however, they were rapidly degraded (t$\sb{1/2} < 2$ min). In the presence hog liver carboxylate esterase, they were converted quantitatively to the corresponding phosphodiesters, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP; after 24 h incubation, only trace amounts of FdUMP, ddUMP, and AZTMP (1-5%) were observed indicating that the PIV$\sb1$ compounds were poor substrates for the enzyme. In human plasma, the PIV$\sb2$ compounds were rapidly degraded with half-lives of less than 5 min. The rate of degradation of the PIV$\sb2$ compounds in the presence of phosphodiesterase I was the same as that in buffer controls, indicating that they were not substrates for this enzyme. In the presence of phosphodiesterase I, PIV$\sb1$-FdUMP, PIV$\sb1$-ddUMP, and PIV$\sb1$-AZTMP were converted quantitatively to FdUMP, ddUMP, and AZTMP.^ PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were effective at controlling HIV type 1 infection in MT-4 and CEM tk$\sp-$ cells in culture. Mechanistic studies demonstrated that PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP were taken up by the cells and converted to ddUTP and AZTTP, both potent inhibitors of HIV reverse transcriptase. However, a potential shortcoming of PIV$\sb2$-ddUMP and PIV$\sb2$-AZTMP as clinical therapeutic agents is that they are rapidly degraded (t$\sb{1/2}$ = approx. 4 minutes) in human plasma by carboxylate esterases. To circumvent this limitation, chemically-labile nucleotide prodrugs and liposome-encapsulated nucleotide prodrugs were investigated. In the former approach, the protective groups bis(N, N-(dimethyl)carbamoyloxymethyl) (DM$\sb2$) and bis (N-(piperidino)carbamoyloxymethyl) (DP$\sb2$) were used to synthesize DM$\sb2$-ddUMP and DP$\sb2$-ddUMP, respectively. In aqueous buffers (pH range 1.0-9.0) these compounds were degraded with half-lives of 3 to 4 h. They had similar half-lives in human plasma demonstrating that they were resistant to esterase-mediated cleavage. However, neither compound gave rise to significant concentrations of ddUMP in CEM or CEM tk$\sp-$ cells. In the liposome-encapsulated nucleotide prodrug approach, three different liposomal formulations of PIV$\sb2$-ddUMP (L-PIV$\sb2$-ddUMP) were investigated. The half-lifes of these L-PIV$\sb2$-ddUMP preparations in human plasma were 2 h compared with 4 min for the free drug. The preparations were more effective at controlling HIV-1 infection than free PIV$\sb2$-ddUMP in human T cells in culture. Collectively, these data indicate that PIV$\sb2$-FdUMP, PIV$\sb2$-ddUMP, and PIV$\sb2$-AZTMP are effective membrane-permeable prodrugs of FdUMP, ddUMP, and AZTMP. ^

HIV-related malignancies: A community-based study using a linkage of cancer registry and HIV registry data

Background. Increased incidence of cancer is documented in immunosuppressed transplant patients. Likewise, as survival increases for persons infected with the Human Immunodeficiency Virus (HIV), we expect their incidence of cancer to increase. The objective of this study was to examine the current gender specific spectrum of cancer in an HIV infected cohort (especially malignancies not currently associated with Acquired Immunodeficiency Syndrome (AIDS)) in relation to the general population.^ Methods. Cancer incidence data was collected for residents of Harris County, Texas who were diagnosed with a malignancy between 1975 and 1994. This data was linked to HIV/AIDS registry data to identify malignancies in an HIV infected cohort of 14,986 persons. A standardized incidence ratio (SIR) analysis was used to compare incidence of cancer in this cohort to that in the general population. Risk factors such as mode of HIV infection, age, race and gender, were evaluated for contribution to the development of cancer within the HIV cohort, using Cox regression techniques.^ Findings. Of those in the HIV infected cohort, 2289 persons (15%) were identified as having one or more malignancies. The linkage identified 29.5% of these malignancies (males 28.7% females 60.9%). HIV infected men and women had incidences of cancer that were 16.7 (16.1, 17.3) and 2.9 (2.3, 3.7) times that expected for the general population of Harris County, Texas, adjusting for age. Significant SIR's were observed for the AIDS-defining malignancies of Kaposi's sarcoma, non-Hodgkin's lymphoma, primary lymphoma of the brain and cancer of the cervix. Additionally, significant SIR's for non-melanotic skin cancer in males, 6.9 (4.8, 9.5) and colon cancer in females, 4.0 (1.1, 10.2) were detected. Among the HIV infected cohort, race/ethnicity of White (relative risk 2.4 with 95% confidence intervals 2.0, 2.8) or Spanish Surname, 2.2 (1.9, 2.7) and an infection route of male to male sex, with, 3.0 (1.9, 4.9) or without, 3.4 (2.1, 5.5) intravenous drug use, increased the risk of having a diagnosis of an incident cancer.^ Interpretation. There appears to be an increased risk of developing cancer if infected with the HIV. In addition to the malignancies routinely associated with HIV infection, there appears to be an increased risk of being diagnosed with non-melanotic skin cancer in males and colon cancer in females. ^

A quality of life subscale for terminally ill cancer patients

A subscale was developed to assess the quality of life of cancer patients with a life expectancy of six months or less. Phase I of this study identified the major concerns of 74 terminally ill cancer patients (19 with breast cancer, 19 with lung cancer, 18 with colorectal cancer, 9 with renal cell cancer, 9 with prostate cancer), 39 family caregivers, and 20 health care professionals. Patients interviewed were being treated at the University of Texas M. D. Anderson Cancer Center or at the Hospice at the Texas Medical Center in Houston. In Phase II, 120 patients (30 with breast cancer, 30 with lung cancer, 30 with colorectal cancer, 15 with prostate cancer, and 15 with renal cell cancer) rated the importance of these concerns for quality of life. Items retained for the subscale were rated as "extremely important" or "very important" by at least 60% of the sample and were reported as being applicable by at least two-thirds of the sample. The 61 concerns that were identified were formatted as a questionnaire for Phase III. In Phase III, 356 patients (89 with breast cancer, 88 with lung cancer, 88 with colorectal cancer, 44 with prostate cancer, and 47 with renal cell cancer) were interviewed to determine the subscale's reliability and sensitivity to change in clinical status. Both factor analysis and item response theory supported the inclusion of the same 35 items for the subscale. Internal consistency reliability was moderate to high for the subscale's domains: spiritual (0.87), existential (0.76), medical care (0.68), symptoms (0.67), social/family (0.66), and emotional (0.61). Test-retest correlation coefficients also were high for the domains: social/family (0.86), emotional (0.83), medical care (0.83), spiritual (0.75), existential (0.75), and symptoms (0.81).^ In addition, concurrent validity was supported by the high correlation between the subscale's symptom domain and symptom items from the European Organization for Research and Treatment of Cancer (EORTC) scale (r = 0.74). Patients' functional status was assessed with the Eastern Cooperative Oncology Group (ECOG) Performance status rating. When ECOG categories were compared to subscale domains, patients who scored lower in functional status had lower scores in the spiritual, existential, social/family, and emotional domains. Patients who scored lower in physical well-being had higher scores in the symptom domain. Patient scores in the medical care domain were similar for each ECOG category. The results of this study support the subscale's use in assessing quality of life and the outcomes of palliative treatment for cancer patients in their last six months of life. ^