EPSTEIN-BARR VIRUS TRANSFORMATION OF B LYMPHOCYTE SUBPOPULATIONS

Epstein-Barr virus is a herpes virus distinguished by its remarkable specificity for the B lymphocyte of humans and certain other primates. Although the transformation process is very efficient, is has become clear that only a fraction of B lymphocytes is susceptible. Therefore the question may be raised if transformation is related to B cell stage of activation. B cells were purified from peripheral blood mononuclear cells by the removal of monocytes using elutriation and sheep red blood cell rosetting to remove T cells. Retesting B cells were purified using discontinuous Percoll gradients. Activation of resting cells for 24 hours with anti-mu or Staphylococcus aureus Cowan I (SAC) resulted in transition of susceptible cells into the G(,1) phase of the cell cycle as shown by an increase in cell size, an increase in uridine incorporation and an increase in sensitivity to B cell growth factor (BCGF). Entry into S phase was achieved by extending the period of activation to 48-96 hr as shown by an increase in thymidine incorporation. By this criterion, SAC activated cells entered S phase on day 2 and anti-mu treated cells on day 3. Control (G(,0)) cells and cells activated for varying lengths of time (G(,1), G(,1) plus S) were exposed to EBV and plated in a limiting dilution assay to determine the frequency of EBV-transformable cells. Control cells and cells activated for 24 hr had a precursor frequency of 1% to 2%. With continued activation, however, precursor frequency decreased as a function of the duration of activation. The decrease in frequency of transformable cells correlated with the entry of the population into S phase. The transformation frequency in the SAC-treated population was reduced twenty-fold on day 4, whereas in the anti-mu treated population it was reduced ten-fold. Treating cells with BCGF in conjunction with low concentrations of anti-mu decreased the transformation frequency to levels lower than anti-mu alone, further suggesting that entry into S phase is accompanied by a reduction in transformability. These results indicate that resting B cells are highly susceptible to transformation and that with in vitro activation into the cell cycle B cells become progressively insensitive to EBV. ^

A PHARMACOLOGICAL EVALUATION OF THE MECHANISM OF CYTOTOXICITY OF 9-BETA-D- XYLOFURANOSYLADENINE IN CHINESE HAMSTER OVARY CELLS

The biochemical determinants of cytotoxicity of the purine nucleoside analog, 9-(beta)-D-xylofuranosyladenine (xyl-A) were studied in wild-type Chinese hamster ovary cells and in nucleoside kinase deficient mutants. It was found that {('3)H}xyl-A was readily phosphorylated to the triphosphate level in both the wild-type and deoxycytidine kinase deficient mutant, but not by the adenosine kinase deficient cells. Values for the apparent Km and Vmax of this uptake process were 43.9 (mu)M and 118.7 nmol/min/10('9) cells, respectively. Cloning procedures indicated that the viability of CHO cells was decreased 90 per cent by a 5-hr incubation with 10 (mu)M xyl-A. However, the toxicity of xyl-A was increased 100-fold by the addition of a nontoxic concentration (10 (mu)M) of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to the medium. High-pressure liquid chromatographic analysis indicated that after 5 hr, the concentration of 9-(beta)-D-xylofuranosyladenine 5'-triphosphate (xyl-ATP) in cells incubated with xyl-A plus EHNA was 2.0 mM, four times greater than in those cells incubated with xyl-A alone. Incubation with xyl-A plus EHNA had no significant effect on the cellular concentrations of 5-phosphoribosyl-1-pyrophosphate after 1 hr whereas, treatment with 3'-dexoyadenosine (cordycepin) decreased the concentration of this metabolite. Determinations of the cellular nucleoside triphosphates indicated that under conditions that resulted in an intracellular accumulation of 500 (mu)M xyl-ATP, the endogenous concentrations of neither the ribonucleoside triphosphates nor deoxyribonucleoside triphosphates were significantly different from those of control cells. The ID(,50) for {('3)H}thymidine incorporation into DNA, 105 (mu)M xyl-ATP, was four-fold less than the ID(,50) for {('3)H}uridine incorporation into RNA suggesting that the process of DNA synthesis is more sensitive to the presence of xyl-ATP. When removed from exogenous xyl-A, CHO cells failed to recover their ability to synthesize RNA and DNA, although the intracellular xyl-ATP concentration decreased to less than 35 (mu)M. The selective inhibition of RNA synthesis by 6-azauridine did not prevent the expression of toxicity by xyl-ATP. However, the selective inhibition of DNA synthesis by ara-C significantly spared toxicity in cells that had accumulated an otherwise lethal concentration of xyl-ATP. It is shown that in cells which had accumulated 1.27 mM {('3)H}xyl-ATP, {('3)H}xyl-A was found to terminate cellular RNA chains at a frequency of 1.42 (mu)mol of {('3)H}xyl-A 3' termini per mol of mononucleotide. These results indicate that a general mechanism for the toxicity of xyl-A to CHO cells includes the cellular accumulation of xyl-ATP, which serves as a substrate for RNA synthesizing enzymes and subsequently is incorporated into nascent RNA transcripts as a chain terminator. A specific mechanism involving the premature termination of RNA primers required for the initiation of DNA synthesis is proposed to account for the inhibitory action of xyl-ATP on DNA synthesis. ^

UPTAKE, METABOLISM, AND METABOLIC EFFECTS OF THE ANTITUMOR TRICYCLIC NUCLEOSIDE, 3-AMINO-1, 5-DIHYDRO-5-METHYL-1-BETA-D-RIBOFURANOSYL-1, 4, 5, 6, 8 PENTAAZAACENAPHTHYLENE

The uptake, metabolism, and metabolic effects of the antitumor tricyclic nucleoside (TCN, NSC-154020) were studied in vitro. Uptake of TCN by human erythrocytes was concentrative, resulting mainly from the rapid intracellular phosphorylation of TCN. At high TCN doses, however, unchanged TCN was also concentrated within the erythrocytes. The initial linear rate of TCN uptake was saturable and obeyed Michaelis-Menten kinetics. TCN was metabolized chiefly to its 5'-monophosphate not only by human erythrocytes but also by wild-type Chinese hamster ovary (CHO) cells. In addition, three other metabolites were detected by means of high-performance liquid chromatography. The structures of these metabolites were elucidated by ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and further confirmed by incubations with catabolic enzymes and intact wild-type or variant CHO cells. All were novel types of oxidative degradation products of TCN. Two are proposed to be (alpha) and (beta) anomers of a D-ribofuranosyl nucleoside with a pyrimido{4,5-c}pyridazine-4-one base structure. The third metabolite is most likely the 5'-monophosphate of the (beta) anomer. A CHO cell line deficient in adenosine kinase activity failed to phosphorylate either TCN or the (beta) anomer. No further phosphorylation of the 5'-monophosphates by normal cells occurred. Although the pathways leading to the formation of these TCN metabolites have not been proven, a mechanism is proposed to account for the above observations. The same adenosine kinase-deficient CHO cells were resistant to 500 (mu)M TCN, while wild-type cells could not clone in the presence of 20 (mu)M TCN. Simultaneous addition of purines, pyrimidines, and purine precursors failed to reverse this toxicity. TCN-treatment strongly inhibited formate or glycine incorporation into ATP and GTP of wild-type CHO cells. Hypoxanthine incorporation inhibited to a lesser degree, with the inhibition of incorporation into GTP being more pronounced. Although precursor incorporation into GTP was inhibited, GTP concentrations were elevated rather than reduced after 4-hr incubations with 20 (mu)M or 50 (mu)M TCN. These results suggested an impairment of GTP utilization. TCN (50 (mu)M) inhibited leucine and thymidine incorporation into HClO(,4)-insoluble material to 30-35% of control throughout 5-hr incubations. Incorporation of five other amino acids was inhibited to the same extent as leucine. Pulse-labeling assays (45 min) with uridine, leucine, and thymidine failed to reveal selective inhibition of DNA or protein synthesis by 0.05-50 (mu)M TCN; however, the patterns of inhibition were similar to those of known protein synthesis inhibitors. TCN 5'-monophosphate inhibited leucine incorporation by rabbit reticulocyte lysates; the inhibition was 2000 times less potent than that of cycloheximide. The 5'-monophosphate failed to inhibit a crude nuclear DNA-synthesizing system. Although TCN 5'-monophosphate apparently inhibits purine synthesis de novo, its cytotoxicity is not reversed by exogenous purines. Consequently, another mechanism such as direct inhibition of protein synthesis is probably a primary mechanism of toxicity. ^

INHIBITION OF THE MITOCHONDRIAL RESPIRATION OF TUMOR CELLS BY SOLUBLE FACTORS RELEASED BY ACTIVATED MACROPHAGES (CYTOTOXICITY, ELECTRON TRANSPORT, MONOCYTE)

Particular interest has been directed towards the macrophage as a primary antineoplastic cell due to its tumoricidal properties in vitro and the observation that an inverse relationship exists between the number of macrophages infiltrating a tumor and metastatic potential. The mechanism of macrophage-mediated injury of tumor cells remains unknown. Recently, it has been shown that injured tumor cells have defective mitochondrial respiration. Our studies have shown that activated macrophages can release soluble factors which can alter tumor cell respiration.^ The effects of a conditioned supernatant (CS) from cultures of activated macrophages on tumor cell (TC) mitochondrial respiration was studied. CS was obtained by incubation of BCG-elicited, murine peritoneal macrophage with RPMI-1640 supplemented with 10% FCS and 50 ng/ml bacterial endotoxin. This CS was used to treat cultures of EMT-6 TC for 24 hours. Mitochondrial respiration was measured polarigraphically using a Clark-type oxygen electrode. Cell growth rate was assessed by ('3)H-Thymidine incorporation. Exposure of EMT-6 TC to CS resulted in the inhibition of malate and succinate oxidation 76.6% and 72.9%, respectively. While cytochrome oxidase activity was decreased 61.1%. This inhibition was accompanied by a 98.8% inhibition of DNA synthesis (('3)H-Thymidine incorporation). Inhibition was dose-related with a 21.3% inhibition of succinate oxidase from a 0.3 ml dose of CS and a 50% inhibition with 1.0 mls. Chromatography of CS on Sephacryl S-200 resulted in isolation of an 80,000 and a 55,000 dalton component which contained the respiration inhibiting activity (RIF). These factors were distinct from a 120,000 dalton cytolytic factor determined by bioassay on Actinomycin-D treated L929 cells. RIF activity was also distinct from several other cytostatic factors but was itself associated with 2 peaks of cytostatic activity. Characterization of the RIF activity showed that it was destroyed by trypsin and heat (100(DEGREES)C, 5 min). It was stable over a broad range of pH (4-9) and its production was inhibited by cycloheximide. The RIF did not have a direct effect on isolated mitochondria of TC nor did it induce the formation of a stable intracellular toxin for mitochondria.^ In conclusion, activated macrophages synthesize and secrete an 80,000 and a 55,000 dalton protein which inhibits the mitochondrial metabolism of TC. These factors induce a cytostatic but not a cytolytic effect on TC.^ The macrophage plays a role in the control of normal and tumor cell growth and in tissue involution. Inhibition of respiration may be one mechanism used by macrophages to control cell growth.^

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

TEMPORAL VARIATION OF ACUTE AND LUNG TUMORIGENIC EFFECTS OF URETHAN ON STRAIN A MICE (CHRONOBIOLOGY, CARCINOGENICITY, ANESTHETIC, DNA SYNTHESIS)

The effect of circadian variation on susceptibility to the chemical induction of cancer was assessed utilizing the mouse pulmonary adenoma bioassay. Different groups of male A/Jax mice (standardized for rhythm analysis with light from 0600-1800 and darkness from 1800-0600) each received a single timed i.p. injection of urethan (Bioassay I: 0.25, 0.5 or 1.0 mg/g body weight; Bioassay II: 0.75, 1.0, 1.25 mg/g body weight; Bioassay III: 1.0 mg/g body weight) at the following times, 0100, 0500, 0900, 1300, 1700 or 2100. Mice were sacrificed 16 weeks after treatment. The tumorigenic effect of urethan on the lungs (lung surface pulmonary adenomas) was assessed. In addition, mortality, body weight changes and the anesthetic effect of urethan were determined. The rhythmic pattern of DNA synthesis in the lung and the comparative rhythmic pattern in the liver were assessed using a tritiated thymidine incorporation assay.^ In the first adenoma bioassay, the lung tumorigenic response in mice given the highest dose of urethan exhibited a 12-hour rhythm with a major peak in tumor yield at 0100 and a secondary peak at 1300; reduced yields occurred at 0500-0900 and 2100. The second adenoma bioassay, studied at a 6-month seasonal divergence in time from the first study showed a peak at 1300 but not at 0100. The mice from the third adenoma bioassay, studied at an 11-month seasonal divergence in time from the 2nd study showed an increase in tumor yield during the rest cycle (0900-1700).^ This study found a definite suggestion of a low amplitude rhythm in susceptibility to urethan induced effects. The acute toxic and pharmacological effects correlated to exhibit a maximal effect during dark hours (activity span). This rhythmicity might be explained by an alteration in the amplitude of hepatic metabolism. The chronic carcinogenic response exhibited an opposite pattern. Urethan induced tumor response was greater during daylight hours (rest cycle). This correlated with the slight elevation in DNA synthetic activity found in the lung and liver which might be responsible for the increase in carcinogenic response. (Abstract shortened with permission of author.) ^

In vitro incorporation of molybdate into demolybdoproteins in Escherichia coli.

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.

Gene therapy of cancer: Mechanisms of ``bystander effect'' killing in cells expressing the herpes simplex virus-thymidine kinase gene and its potential clinical applications

At the fore-front of cancer research, gene therapy offers the potential to either promote cell death or alter the behavior of tumor-cells. One example makes use of a toxic phenotype generated by the prodrug metabolizing gene, thymidine kinase (HSVtk) from the Herpes Simplex Virus. This gene confers selective toxicity to a relatively nontoxic prodrug, ganciclovir (GCV). Tumor cells transduced with the HSVtk gene are sensitive to 1-50 $\mu$M GCV; normal tissue is insensitive up to 150-250 $\mu$M GCV. Utilizing these different sensitivities, it is possible to selectively ablate tumor cells expressing this gene. Interestingly, if a HSVtk$\sp+$ expressing population is mixed with a HSVtk$\sp-$ population at high density, all the cells are killed after GCV administration. This phenomenon for killing all neighboring cells is termed the "bystander effect", which is well documented in HSVtk$\sp-$ GCV systems, though its exact mechanism of action is unclear.^ Using the mouse colon carcinoma cell line CT26, data are presented supporting possible mechanisms of "bystander effect" killing of neighboring CT26-tk$\sp-$cells. A major requirement for bystander killing is the prodrug GCV: as dead or dying CT26tk$\sp+$ cells have no toxic effect on neighboring cells in its absence. In vitro, it appears the bystander effect is due to transfer of toxic GCV-metabolites, through verapamil sensitive intracellular-junctions. Additionally, possible transfer of the HSVtk enzyme to bystander cells after GCV addition, may play a role in bystander killing. A nude mouse model suggests that in a 50/50 (tk$\sp+$/tk$\sp-$) mixture of CT26 cells the bystander eradication of tumors does not involve an immune component. Additionally in a possible clinical application, the "bystander effect" can be directly exploited to eradicate preexisting CT26 colon carcinomas in mice by intratumoral implantation of viable or lethally irradiated CT26tk$\sp+$ cells and subsequent GCV administration. Lastly, an application of this toxic phenotype gene to a clinical marking protocol utilizing a recombinant adenoviral vector carrying the bifunctional protein GAL-TEK to eradicate spontaneously-arisen or vaccine-induced fibrosarcomas in cats is demonstrated. ^

Hispanic incorporation and access to health care in Houston and Harris County, Texas: Adopting an Hispanic perspective to the Aday and Andersen model

This study examines Hispanic levels of incorporation and access to health care. Applying the Aday and Andersen framework for the study of access, the study examined the relationship between two levels of Hispanic incorporation into U.S. society, i.e., mainstream versus ethnic, and potential and realized measures of access to health care. Data for the study were drawn from a 1992 telephone survey of 600 randomly selected Hispanics in Houston and Harris County.^ The hypotheses tested were: (1) Hispanics who are incorporated into mainstream society are more likely to have better potential and realized access to health care than those who are incorporated into ethnic-group enclaves regardless of their socioeconomic status (SES), health status and health needs, and (2) there is no interaction between the levels of incorporation (mainstream or ethnic) and SES, health status, and health needs in predicting potential and realized access.^ The data analysis supported Hypothesis One for the two measures of potential access. The results of bivariate and multiple logistic regression analyses indicated that for Hispanics in Houston and Harris County, being in the "mainstream" incorporation category increased their potential access to care, having "health insurance" and a "regular place of care". For the selected measure of realized access, having a "regular check-up", the analysis did not demonstrate statistically significant differences in having a regular check-up among Hispanics incorporated in the ethnic or mainstream incorporation categories.^ Hypothesis Two, that there is no interaction between the levels of incorporation and socioeconomic characteristics, health status, and health needs in predicting potential and realized access among Hispanics was supported by the data. The results of the logistic regression analysis showed that, after adjusting for socioeconomic status, health status, and health needs, the association between "level of incorporation" and the two measures of potential access ("health insurance" and having a "usual place of care") was not modified by the control variables nor by their interaction with level of incorporation. That is, the effect of incorporation on Hispanics' health insurance coverage, and having a usual place of care, was homogenous across Hispanics with different SES and health status.^ The main research implication of this dissertation is the employment of a theoretical framework for the assessment of cultural factors essential to research on migrating heterogeneous subpopulations. It also provided strategies to solve practical and methodological difficulties in the secondary analyses of data on these populations. ^