Metabolism and action of 9-$\beta$-D-arabinofuranosyl-2-fluoroadenine

9-$\beta$-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and 2$\sp\prime$-deoxyadenosine with potent antitumor activity both in vitro and in vivo. The mechanism of action of F-ara-A was evaluated both in whole cells and in experimental systems with purified enzymes. F-ara-A was converted to its 5$\sp\prime$-triphosphate F-ara-ATP in cells and then incorporated into DNA in a self-limiting manner. About 98% of the incorporated F-ara-AMP residues were located at the 3$\sp\prime$-termini of DNA strands, suggesting a chain termination property of this compound. DNA synthesis in CEM cells was inhibited by F-ara-A treatment with an IC$\sb{50}$ value of 1 $\mu$M. Cells were not able to restore the normal level of DNA synthesis even after being cultured in drug-free medium for 40 h. A DNA primer extension assay with M13mp18(+) single-stranded DNA template using purified human DNA polymerases $\alpha$ and further revealed that F-ara-ATP competed with dATP for incorporation into the A sites of the elongating DNA strands. The incorporation of F-ara-AMP into DNA resulted in a termination of DNA synthesis at the incorporated A sites. Pol $\alpha$ and $\delta$ were not able to efficiently extend the DNA primer with F-ara-AMP at its 3$\sp\prime$-end. Furthermore, the presence of F-ara-AMP at the 3$\sp\prime$-end of an oligodeoxyribonucleotide impaired its ligation with an adjacent DNA fragment by human and T4 ligases. Human DNA polymerase $\alpha$ incorporated more F-ara-AMP into DNA than polymerase $\delta$ and was more sensitive to the inhibition by F-ara-ATP, suggesting that polymerase $\alpha$ may be a preferred target for this analogue. On the other hand, DNA-dependent nucleotide turnover experiments and sequencing gel analysis demonstrated that DNA polymerase $\delta$ was able to remove the incorporated F-ara-AMP residue from the 3$\sp\prime$-end of the DNA strand with its 3$\sp\prime$-5$\sp\prime$ exonuclease activity in vitro, subsequently permitting further elongation of the DNA strand.^ The incorporation of F-ara-AMP into DNA was linearly correlated both with the inhibition of DNA synthesis and with the loss of clonogenicity. Termination of DNA synthesis and deletion of genetic material resulted from F-ara-AMP incorporation may be the mechanism responsible for cytotoxicity of F-ara-A. (Abstract shortened with permission of author.) ^

Studies of the mechanism of daunorubicin carcinogenesis: Modulation by vitamin E

Daunorubicin (DNR) is an anthracycline antibiotic used as a cancer chemotherapeutic agent. However, it causes mammary adenocarcinomas in female Sprague-Dawley (SD) rats. Vitamin E (E) has been found to reduce DNR carcinogenicity. I investigated the mechanism of DNR carcinogenicity and its interaction with E in SD rats by studying DNR-DNA adduct formation and the influence of E status on DNR clearance and free radical producing and detoxifying enzymes.^ The hypothesis was that DNR exerts its tumorigenic effect via free radicals generated during redox cycling and production of reactive intermediates capable of forming DNA adducts. E was postulated to act as a protective agent through a combination of its antioxidant property, modulation of drug clearance and levels of free radical producing and detoxifying enzymes.^ DNA adduct formation was measured by the nuclease P1 $\sp{32}$P-post labeling assay. In vitro, DNR was activated by rat liver microsomes and either NADPH or cumene hydrogen peroxide (CuOOH). Rat liver DNA incubated with this mixture formed two adducts when the cofactor was NADPH and three adducts when CuOOH was used. In vivo, SD rats were treated with i.v. doses of DNR. No detectable DNR-DNA adducts were formed in liver or mammary DNA in vivo, although there was an intensification of endogenous DNA adducts.^ Groups, 1, 2, 3 and 4 of weanling female SD rats were fed 0, 100, 1,000 and 10,000 mg $\alpha$-tocopheryl acetate/kg diet respectively. A comparison of Groups 1 and 4 showed no effect of E status on clearance of 10 mg tritiated DNR/kg body weight over 72 hours. However, liver cleared DNR at a faster rate than mammary epithelial cells (MEC).^ Xanthine oxidase, which catalyzes DNR redox cycling, was significantly decreased in liver and MEC of rats in group 4 compared to groups 1, 2, and 3. Detoxifying enzymes were not dramatically affected by E supplementation. Quinone reductase in MEC was significantly increased in group 4 compared to other groups. Overall, the liver had higher levels of free radical detoxifying enzymes compared to MEC.^ These data support a role of free radicals in DNR carcinogenicity because (1) endogenous DNA adducts formed due to free radical insult are further intensified by DNR treatment in vivo, (2) MEC, the specific target of DNR carcinogenicity, cannot rapidly clear DNR and have a lower free radical detoxifying capability than liver, (3) E supplementation caused lowering of free radical generating potential via xanthine oxidase, and increased DNR detoxification due to elevation of quinone reductase in MEC. ^

Cancer patients' attitudes toward cancer trials

Background. Accurate measurement of attitudes toward participation in cancer treatment trials (CTs) and cancer prevention trials (CPTs) across varied groups could assist health researchers and educators when addressing attitudinal barriers to participation in these trials. ^ Methods. The Attitudes toward Cancer Trials Scales (ACTS) instrument development was based on a conceptual model developed from research literature, clinical practice experience, and empirical testing of items with a sample of 312 respondents. The ACTS contains two scales, the Cancer Trials (CT) scale (4 components; 18 items) and the Cancer Prevention Trials (CPT) scale (3 components; 16 items). Cronbach's alpha values for the CT and CPT scales, respectively, were 0.86 and 0.89. These two scales along with sociodemographic and cancer trial history variables were distributed in a mail survey of former patients of a large cancer research center. The disproportionate stratified probability sampling procedure yielded 925 usable responses (54% response rate). ^ Results. Prevalence of favorable attitudes toward CTs and CPTs was 66% and 69%, respectively. There were no significant differences in mean scale scores by cancer site or gender, but African Americans had more favorable attitudes toward CTs than European Americans. Multiple regression analysis indicated that older age, lower education level, and prior CT participation history were associated with more favorable attitudes toward CTs. Prior CT participation and prior CPT participation were associated with more favorable attitudes toward CPTs. Results also provided evidence of reliability and construct validity for both scales. ^ Conclusions. Middle age, higher education, and European American ethnicity are associated with less positive attitudes about participating in cancer treatment trials. Availability of a psychometrically sound instrument to measure attitudes may facilitate a better understanding decision making regarding participation in CTs and CPTs. It is this author's intention that the ACTS' scales will be used by other investigators to measure attitudes toward CTs and CPTs in various groups of persons, and that the many issues regarding participation in trials might become more explicit. ^

Studies on the adhesion properties of the liver-metastatic murine RAW117 large-cell lymphoma cells: Roles of $\beta\sb3$ integrin

Metastasis is the major cause of death in cancer patients. Since many cancers show organ-preference of metastasis, elucidation of the underlying mechanisms of metastasis will benefit diagnosis or treatment of metastatic diseases. Adhesion mechanisms are thought to be involved in organ-preference of metastasis, because metastatic cells show organ preference in adhering to organ-derived microvascular endothelial cells. The adhesion molecules in this process remain largely unidentified. I have examined a series of murine RAW117 large-cell lymphoma cells variants selected in vivo for liver-colonizing properties ($\rm{H10{>>}L17>P}$). The highly liver-metastatic H10 cells were found to differentially express much higher levels of integrin $\alpha\rm\sb{v}\beta\sb3$ than L17 or P cells. H10 cells also adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin and type I collagen, and adhered at significantly higher rates to (GRGDS)$\sb4$ than to monomeric RGD-peptides. In contrast, P and L17 cells did not adhere well to the above substrates. H10 cells also spread well on vitronectin and migrated toward vitronectin concentration gradients. Pretreament of H10 cells with anti-$\beta\sb3$ monoclonal antibodies resulted in significant decreases in adhesion of H10 cells to vitronectin and immobilized (GRGDS)$\sb4$, and reduced the formation of experimental liver metastases in syngeneic Balb/c mice.^ Adhesion of RAW117 cells under hydrodynamic shear stresses was also studied because tumor cell adhesion occurs under fluid shear stresses in target organ microvessels. Similar to their properties found with static adhesion assays, H10 cells stabilized their hydrodynamic adhesion to vitronectin, fibronectin and (GRGDS)$\sb4$ much more quickly than P or L17 cells. Unlike their static adhesion properties, RAW117 cells showed differential adhesion stabilization to liver-sinusoidal endothelial cell-derived extracellular matrix ($\rm{H10{>>}L17>P}$). Although not supporting static adhesion of RAW117 cells, monomeric RGD-peptides mediated adhesion stabilization of H10 cells but not L17 or P cells. Integrin $\rm\alpha\sb{v}\beta\sb3$ was found to be involved in stabilizing H10 cell adhesion to vitronectin, (GRGDS)$\sb4$, monomeric RGD-peptide R1, and liver sinusoidal endothelial cell-derived extracellular matrix.^ This study is the first to provide evidence that integrin $\rm\alpha\sb{v}\beta\sb3$ is differentially expressed in liver-metastatic lymphoma cells and involved in differential adhesion of these cells. The results indicate that strong static adhesion and especially the unique hydrodynamic adhesion of RAW117 cells to the RGD-containing substrates correlate with liver-metastatic potentials. Thus, integrin $\rm\alpha\sb{v}\beta\sb3$ may play an important role in liver-preferential metastasis of RAW117 large-cell lymphoma cells. ^

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. ^

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. ^

The volume effect in irradiated mouse colorectum

Damage of the colorectum is the dose-limiting normal tissue complication following radiotherapy of prostate and cervical cancers. One approach for decreasing complications is to physically reduce the treatment volume. Mathematical models have been previously developed to describe the change in associated toxicity with a change in irradiated volume, i.e. the "volume effect", for serial-type normal tissues including the colorectum. The first goal of this thesis was to test the hypothesis that there would not be a threshold length in the development of obstruction after irradiation of mouse colorectum, as predicted by the Probability model of the volume effect. The second goal was to examine if there were differences in the threshold and in the incidence of colorectal obstruction after irradiation of two mouse strains, C57B1/6 (C57) and C3Hf/Kam (C3H), previously found to be fibrosis-prone and-resistant, respectively, after lung irradiation due, in part, to genetic differences. The hypothesis examined was that differences in incidence between strains were due to the differential expression of the fibrogenic cytokines $\rm TGF\beta$ and $\rm TNF\alpha.$ Various lengths of C57 and C3H mouse colorectum were irradiated and the incidence of colorectal obstruction was followed up to 15 months. A threshold length was observed for both mouse strains, in contradiction of model predictions. The mechanism of the threshold was epithelial regeneration after irradiation. C57 mice had significantly higher incidence of colorectal obstruction compared to C3H mice, especially at smaller irradiated lengths. Colorectal tissue was obtained at various times after irradiation and prepared for histology, immunohistochemistry and RNase protection assay for measurement of $\rm TGF\beta 1,$ 2, 3 and $\rm TNF\alpha$ mRNA. Distinct strain differences in the histological time of appearance and spatial locations of fibrosis were observed. However, there were no consistent strain difference in mRNA levels or immunolocalization for any of the cytokines examined. The data indicate the need for volume effect models that account for biologically important processes, such as the effect of epithelial regeneration after irradiation. As well, changes in fibrogenic cytokines at the mRNA level do not contribute to the strain difference in radiation-induced colorectal obstruction. ^

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.) ^

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.) ^

Evidence for the involvement of proximal chromosome 3P loci in the progression of Von Hippel-Lindau related tumors

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of retinal and central nervous system hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic islet cell tumors (PICT). The VHL gene maps to chromosome 3p25 and has been shown to be mutated in 57% of sporadic cases of RCC, implicating VHL in the genesis of RCC. We report a multigeneration VHL kindred in which four affected female siblings developed PICT at early ages. Analysis of the three coding exons of the VHL gene in this family revealed a single, missense mutation in codon 238. Inheritance of the 238 mutation has been reported to correlate with a 62% risk of pheochromocytoma development. In this kindred, all affected individuals carried the mutation as well as one additional sibling who showed no evidence of disease. Clinical screening of this individual indicated small ($<$1 cm) pancreatic and kidney tumors. Results suggest that inheritance of the codon 238 mutation does not correlate with early onset pheochromocytoma. Rather, the only individual in the pedigree with pheochromocytoma was the proband's mother who developed bilateral pheochromocytoma at the age of 62. Thus, the VHL codon 238 mutation may predispose to late onset pheochromocytoma in this family; however, it does not explain the preponderance of PICT in the third generation since this mutation has not been reported to increase the risk of developing pancreatic lesions. This suggests that inheritance of the codon 238 mutation and subsequent somatic inactivation of the wild type allele of the VHL gene may not be sufficient to explain the initiation and subsequent progression to malignancy in VHL-associated neoplasms. Since the two tumor types that most frequently progress to malignancy are RCC and PICT, we asked whether loss of heterozygosity (LOH) could be detected proximal to the VHL gene on chromosome 3 in distinct regions of 3p previously implicated by LOH and cytogenetic studies to contain tumor suppressor loci for RCC. LOH was performed on high molecular weight DNA isolated from peripheral blood and frozen tumor tissue of family members using microsatellite markers spanning 3p. Results indicated LOH for all informative 3p loci in tumor tissue from affected individuals with PICT. LOH was detected along the entire length of the chromosome arm and included the proximal region of 3p13-14.2 implicated in the hereditary form of renal cell carcinoma.^ If 3p LOH were a critical event in pancreatic islet cell tumorigenesis, then it should be expected that LOH in sporadic islet cell tumors would also be observed. We expanded LOH studies to include sporadic cases of PICT. Consistent LOH was observed on 3p with a highest frequency LOH in the region 3p21.2. This is the first evidence for an association between chromosome 3 loci and pancreatic islet cell tumorigenesis. (Abstract shortened by UMI.) ^

Genetic analysis of tumor progression susceptibility in the mouse skin model

In the field of chemical carcinogenesis the use of animal models has proved to be a useful tool in dissecting the multistage process of tumor formation. In this regard the outbred SENCAR mouse has been the strain of choice in the analysis of skin carcinogenesis given its high sensitivity to the chemically induced acquisition of premalignant lesions, papillomas, and the later progression of these lesions into squamous cell carcinomas (SCC).^ The derivation of an inbred strain from the SENCAR stock called SSIN, that in spite of a high sensitivity to the development of papillomas lack the ability to transform these premalignant lesions into SCC, suggested that tumor promotion and progression were under the genetic control of different sets of genes.^ In the present study the nature of susceptibility to tumor progression was investigated. Analysis of F1 hybrids between the outbred SENCAR and SSIN mice suggested that there is at least one dominant gene responsible for susceptibility to tumor progression.^ Later development of another inbred strain from the outbred SENCAR stock, that had sensitivity to both tumor promotion and progression, allowed the formulation of a more accurate genetic model. Using this newly derived line, SENCAR B/Pt. and SSIN it was determined that there is one dominant tumor progression susceptibility gene. Linkage analysis showed that this gene maps to mouse chromosome 14 and it was possible to narrow the region to a 16 cM interval.^ In order to better characterize the nature of the progression susceptibility differences between these two strains, their proliferative pattern was investigated. It was found that SENCAR B/Pt, have an enlarged proliferative compartment with overexpression of cyclin D1, p16 and p21. Further studies showed an aberrant overexpression of TGF-$\beta$ in the susceptible strain, an increase in apoptosis, p53 protein accumulation and early loss of connexin 26. These results taken together suggest that papillomas in the SENCAR B/Pt. mice have higher proliferation and may have an increase in genomic instability, these two factors would contribute to a higher sensitivity to tumor progression. ^

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. ^