Streptonigrin is a antineoplastic antibiotic whose mode of action is dependent on formation of the hydroxyl radical. A streptonigrin resistance gene was cloned from a partial Sau3A digest of E. coli DNA. One to 10 Kilobase (Kb) fragments were ligated into the vector pUC18 and transformed into E. coli (DH5{dollar}\alpha{dollar}). Once prepared, the library was replica plated and then screened with differing streptonigrin concentrations in a top agar overlay. Clones exhibiting growth into the overlay at the highest STN concentrations (pH191, pH192) were rescued from the overlay and grown for plasmid isolation and for determination of theminimum inhibitory concentration (MIC) for streptonigrin in LB broth, 3 and 1 {dollar}\mu{dollar}g/ml respectively. The isolated plasmids were mapped by restriction endonuclease digestion followed by agarose gel electrophoresis. Restriction sites were assigned based on known location in the multicloning site of the vector, leading to the production of an approximate physical map of the insert in the plasmid. The resistance element (StnR) was localized by deletion studies on pH191. Sequencing and analysis of the deoxynucleotide sequence of pH191 led to the identification of an open reading frame (StnR) with a high homology (97.6%) to the first 185 residues of riboflavin synthase (RibC). The resistance gene was expressed in S. lividans on pIJ702 where it yielded streptonigrin resistance in excess of that found in the wild type producing species, S. flocculus (40 {dollar}\mu{dollar}g/ml). Expression of StnR and RibC in E. coli via the vector pET3C resulted in strains with elevated streptonigrin resistance as compared to the pET3C control in both solid and liquid media. A gene imparting resistance to the hydroxyl radical producing drug streptonigrin has been identified as the riboflavin synthase from E. coli.

The homeostatic mechanism for the respiratory system involves the tight control of expression of several gene products. One such gene is a member of the multi-substrate oxidase system called cytochrome P450 (P450). Pulmonary P450s may be involved in the tissue-specific detoxification, and/or bio-activation of chemical agents. Additionally, pulmonary cytochrome P450s, particularly the human iso-form 4B1, could play an important role in the development and homeostasis of the pulmonary tissue by removing androgens that cause deleterious effects on lung maturation. Human cytochrome P450 4B1 (CYP 4B1) is a predominant cytochrome P450 activity in the lung. CYP 4B1 protein is reported to bio-activate some pulmonary toxicants. Therefore it may mediate chemical-induced damage to that organ. However, the high expression levels of CYP 4B1 gene products in the lung (approximately 80% of the total P450 activity) may suggest the involvement of the iso-form in normal physiological functions for pulmonary tissue. On the other hand, the results of this and other laboratories have shown an apparent species-specific difference in the catalytic activity of 4B1 protein in man versus rodents. For instance, the rodent iso-form is potent at activating pro-carcinogens and devoid of androgen hydrolyzing activities towards androgen metabolism. The human 4B1 protein has higher 6-beta-testosterone hydroxylase activity while the rodent iso-form is devoid of this action. Furthermore, the manner in which the 4B1 gene is regulated appears to be species-specific. Therefore, my studies have been directed toward further defining the genetic mechanism of action that regulates the human 4B1 gene expression levels. My research objectives have been divided into three specific aims: to determine the presence or absence of cis-acting elements located in the 3.0 Kbp upstream fragment of the human 4B1 gene; to determine the DNA structure of the 4.5 Kbp fragment that overlaps and lies further upstream to the 3.0 Kbp regulatory region of the human 4B1 gene; and to determine the actions of a contiguous 6.5 Kbp fragment containing the entire putative upstream region of 4B1 gene on transcription of the luciferase reporter gene in human lung cells. The results of my studies show the presence of multiple promoter elements in addition to the TATA-box that are contained in the CYP 4B 13.0 Kbp upstream fragment. Nucleotide sequence analysis of the 4.5 Kbp upstream fragment that contains 1.0 Kbp overlapping fragment with the 3.0 Kbp fragment based on restriction mapping confirms that it is indeed part of the contiguous region of the CYP 4B1 gene. A number of consensus DNA regulatory motifs have been identified. (Abstract shortened by UMI.)

The large subunit of ribonucleotide reductase (RR1) encoded by Herpes Simplex Virus Type 1 and 2 (ICP6 and ICP10, respectively) is a chimeric protein consisting of a Ser/Threonine protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase (RR) domain at the carboxy terminus. The findings that the PK domain is present only in HSV RR1 proteins, it is dispensable for ribonucleotide reduction and it functions as immediate-early (IE) protein during HSV infection, suggest that the PK activity plays a significant role in virus pathogenesis. The present work was initiated to confirm the intrinsic nature of ICP10 PK activity and to elucidate its role in HSV-2 infection. In stably transfected eukaryotic cells, ICP10 PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the transmembrane (TM) segment and it was significantly impaired by mutation of the invariant Lys (Lys{dollar}\sp{lcub}176{rcub}{dollar}). Loss of PK activity by Lys{dollar}\sp{lcub}176{rcub}{dollar} mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp29{dollar}\rm\sp{lcub}la1{rcub}{dollar}) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. In cells infected with laboratory and patient isolates of HSV, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and Immunoglobulin G (IgG). Two HSV-2 (G) mutants deleted in the protein kinase or ribonucleotide reductase domains of ICP10 (ICP10{dollar}\Delta{dollar}PK and ICP10{dollar}\Delta{dollar}RR, respectively) were constructed by marker transfer. ICP10{dollar}\Delta{dollar}PK virus lost its intrinsic PK activity but retained its RR activity; ICP10{dollar}\Delta{dollar}RR virus retained its PK activity but lost its RR activity. ICP10{dollar}\Delta{dollar}PK virus does not replicate during the first 10 hrs postinfection (p.i.). However, its titers catch up with those of the wild type virus by 24 hrs p.i.; ICP10{dollar}\Delta{dollar}RR virus replicates as well as the wild type virus in exponentially growing cells but it is significantly impaired for growth in growth-restricted cells. HSV-2 and ICP10{dollar}\Delta{dollar}RR virus produce similar clear plaques but ICP10{dollar}\Delta{dollar}PK virus produces hazy plaques, which under magnification consist of a mixture of lysed and unlysed cells. The studies suggest that (i) ICP10 has intrinsic auto- and transphosphorylating PK activity, (ii) ICP10 PK and RR are functionally dissociable in virus infected cells, (iii) ICP10 PK is required for virus replication during the first 10 hrs p.i. and (iv) ICP10 PK may be involved in cell death in virus infected cells.

Chromosomal DNA replication in animal cells is a complex process. A complete understanding of this process will require information on the organization and the control of the DNA synthetic machinery. To date several mammalian enzymes and protein factors have been shown to be required for DNA replication in vitro. A variety of mechanisms describing how these proteins might function at the mammalian cell replication fork have also been proposed. However, the manner in which these proteins, and those as yet to be identified, associate with one another, as well as their ability to act in concert allowing for the efficient replication of DNA have not been well defined. Over the years evidence for these proteins organizing into a large macromolecule complex to mediate DNA replication has emerged. In order to pursue the hypothesis of multiprotein complex playing a role in mammalian DNA replication, this study was directed towards identifying and characterizing such DNA replication multiprotein complex from murine cells. By using a series of differential centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps, a multiprotein DNA replication complex was isolated and purified from mouse mammary carcinoma cell line (FM3A). A number of key enzymes and protein factors for DNA replication were found to associate with this multiprotein complex including RPA, DNA topoisomerase I and II, DNA polymerase alpha, DNA primase, DNA polymerase delta, PCNA, RF-C, DNA ligase I and DNA helicase. The complex has a 17S sedimentation coefficient as determined by sucrose density gradient analysis. The integrity of the complex is maintained after treatment with detergents, salt, RNase and DNase, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecule components. Most importantly, it was demonstrated that this complex of proteins is fully competent to replicate polyomavirus (PyV) DNA in vitro. Based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex, a model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC). It was also shown that the in vitro PyV DNA replication reaction catalyzed by the Q-Sepharose column purified mouse cell MRC is dependent on polyoma virus DNA replication origin, Py large T-antigen, Mg++, the energy regenerating system, and an exogenously added PyV origin containing DNA molecule. Kinetics analysis of the initiation of in vitro PyV DNA replication mediated by the purified MRC demonstrated that the lag time for initiation of the DNA replication was reduced significantly compared with what has been reported using human cell crude extracts in the SV40 DNA replication system. A study of the cell cycle-dependent regulation of the MRC in terms of its activity and integrity showed that a fully assembled MRC is maintained in both quiescent and proliferating stages of cells. However, the DNA replication-active form of the MRC can only be found in the S-phase cells.

The role of the putative sigma receptor in mediating neuroprotection against glutamate induced neuronal injury was examined in mature cultured rat cortical neurons. With the exception of the sigma1, selective ligand (+)-3-PPP, all of the sigma receptor ligands tested were neuroprotective, preventing glutamate induced morphological changes and increases in LDH release. When corrected for relative sigma versus PCP binding site affinity, it appears that a positive correlation exists between neuroprotective potency and sigma1, site affinity. None of the sigma ligands were neurotoxic when tested alone at concentrations at least 5-30 times their respective neuroprotective EC{dollar}\sb{lcub}50{rcub}{dollar} values. The effect of neuroprotective sigma ligands on the unique calcium responses evoked by glutamate, NMDA, potassium chloride (KCl) and trans-ACPD were investigated to elucidate the mechanism of sigma-mediated neuroprotection. In general, except for (+)-3-PPP all of the sigma ligands studied interfered with glutamate and NMDA induced (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} signaling, but, highly sigma{dollar}\sb1{dollar} selective ligands also lacking substantial PCP binding site affinity (i.e. carbetapentane, DTG and haloperidol) were much less effective in altering calcium influx induced by 80 {dollar}\mu{dollar}M glutamate. In contrast to glutamate, KCl (50 mM) produced changes in (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} which were not neurotoxic to the neurons as measured by LDH release. Sigma ligands which lack substantial PCP site afflinity were very effective in altering KCl induced calcium signaling while the sigma/PCP site ligand (+)-cyclazocine was ineffective or, in the case of (+)-SKF 10047, much less effective. Similar to the effects of sigma ligands on KCl induced calcium dynamics, the sigma selective ligands DTG, haloperidol, (+)-pentazocine, and carbetapentane were very effective in altering intracellular calcium dynamics evoked by trans-ACPD while the sigma/PCP ligand (+)-SKF 10047 was ineffective or, in the case of (+)-cyclazocine, much less effective. Importantly, (+)-3-PPP, a non-neuroprotective sigma selective ligand, actually potentiated the calcium response elicited by trans-ACPD. The ability of sigma ligands applied at maximal neuroprotective concentrations to attenuate receptor and/or voltage-gated changes in calcium dynamics suggests that modulation of neurotoxic (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} plays a significant role in sigma-mediated neuroprotection. The unique modulatory effects of sigma ligands on the buffering of neuronal (Ca{dollar}\sp{lcub}2+{rcub}\rbrack\sb{lcub}\rm i{rcub}{dollar} will likely have numerous therapeutic applications in the treatment of CNS injury and other neurodegenerative disorders.

Our laboratory have designed and synthesized a number of cytochrome P450 17alpha-hydroxylase/C17,20-lyase (P450c17) inhibitors with the aim of inhibiting androgen synthesis and as potential agents to treat prostate cancer. VN/63-1, VN/85-1, VN/87-1, VN/107-1, VN/108-1, and VN/109-1 inhibitor were chosen for study. To select the most potent inhibitors, a new method for evaluating inhibitors of the P450c17 enzyme was designed. Bacteria expressing the P450c17 were used as a source of the enzyme. HPLC was replaced by a rapid radiometric assay, acetic acid releasing assay. This new analytical system will be a powerful tool to accelerate the process of new compound selection. Utilizing this system, I showed that VN/85-1, VN/87-1, and VN/108-1 are the most potent inhibitors of P450c17. The effects of the new inhibitors on androgen dependent LNCaP and androgen independent PC-3 prostatic carcinoma cells proliferation were studied. I have shown that VN/85-1, VN/87-1, and VN/108-1 are the most potent inhibitors of LNCaP cell growth in vitro . VN/85-1 also has weak inhibitory effects against androgen independent PC-3 cells. The effects of azolyl steroids on LNCaP androgen receptor (AR) were determined. VN/63-1, VN/85-1, and VN/87-1 were full antagonists of LNCaP AR. Although, VN/107-1 , VN/108-1, and VN/109-1 were partial agonists of LNCaP AR, all of the evaluated inhibitors were antiandrogens against the wild-type AR. The new inhibitors were also evaluated in vivo. The male SCID mice bearing LNCaP tumor xenografts were utilized for new compounds evaluation. I showed that VN/85-1 and VN/87-1 have potent antitumor effects in this in vivo model. The ability of pregnenolone to stimulate LNCaP cell proliferation was discovered and investigated. I showed that this pregnenolone stimulation is mediated through the mutated AR and can be inhibited by our novel compounds, especially by VN/63-1, VN/85-1, and VN/87-1. This findings suggests that pregnenolone has a significant role in stimulating prostate cancer and that our new compounds by inhibiting not only androgens but also pregnenolone action may be more effective for treating prostate cancer than currently available agents.

We have isolated a multiprotein complex for DNA synthesis, designated the DNA synthesome, from human breast cancer (MDA MB-468) cells, biopsied human breast tumor tissue and xenografts from nude mice injected with the human breast cancer cell line MCF-7. The breast cell DNA synthesome was shown to fully support the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Moreover, our results obtained from a forward mutagenesis assay indicate that the DNA synthesome isolated from malignant breast cells possesses a lower fidelity for DNA replication in vitro than the complex from a nonmalignant breast cell line. The proteins and enzymes found to copurify with the breast cell DNA synthesome include: DNA polymerases alpha, delta, and epsilon, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), DNA ligase, DNA topoisomerases I and II and poly(ADP-ribose) polymerase. To begin to determine the organization of these DNA synthetic proteins within the breast cell DNA synthesome, we performed co-immunoprecipitation experiments with antibodies directed against DNA polymerases alpha, delta and PCNA. We found that DNA polymerases alpha, delta, DNA primase, RF-C and PCNA tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP and several other components interact with the synthesome via an interaction with only PCNA or DNA polymerase alpha. Furthermore, we employed the breast cell DNA synthesome as a model to study the mechanisms of action of two anti-breast cancer agents that target the DNA synthetic process, irinotecan (CPT-11/SN-38) and etoposide (VP-16). We obtained novel data suggesting that both SN-38 and VP-16 stabilized cleavable complexes represent blocks to replication fork progression, as each agent caused an accumulation of short DNA products during synthesome mediated in vitro replication. Overall, our results indicate that breast cancer cells utilize an asymmetric multiprotein complex to mediate DNA synthesis and that utilization of the DNA synthesome as a drug model may provide important new insights into the mechanisms of action of SN-38 and VP-16.

The Chinese hamster lung cell line DC-3F8/A55 has a 4,500-fold increase in resistance to methotrexate over the parental cell line DC-3F. Although DC- 3F8/A55 cells have a 4.5-fold increase in a mutant form of dihydrofolate reductase, this does not fully account for the high level of resistance to methotrexate. The purpose of this study is to determine the molecular basis for the inability of DC-3F8/A55 cells to accumulate methotrexate, and to identify the mechanism of transport of folate compounds in DC-3F8/A55 cells. This work has revealed that DC-3F8/A55 cells harbor a debilitating mutation to the reduced folate carrier gene, resulting in the loss of reduced folate carrier function. A nonsense mutation changes an arginine at amino acid 88 to a STOP codon, resulting in a non-functional protein. The parental cell line DC-3F is heterozygous at this locus, possessing one mutant and one wild-type allele of the RFC gene, thus retaining reduced folate carrier activity. These facts are supported by the kinetics of folate transport in both of these cell lines. The parental cell line DC-3F has a Kt for folinic acid of 10.69 +/- 0.67 muM and for methotrexate of 8.88 +/- 0.82 muM, values characteristic of a cell expressing a reduced folate carrier. DC-3F8/A55 cells were found to have a Kd for MTX of 3.16 +/- 1.03 nM, for folinic acid of 7.75 +/- 2.16 nM, and for folic acid of 1.42 +/- 0.54 nM. The high affinity of DC-3F8/A55 cells for folic acid, with a Kd for folic acid in the nM range, suggests that these cells are expressing a folate receptor. Northern blot analysis revealed a 1.6 kb transcript with low homology to FR-alpha and FR-gamma in DC-3F8/A55 cells. Overall, these studies suggest that the methotrexate transport-defective cell line DC-3F8/A55 expresses a previously unidentified folate receptor which may be a new member of the folate receptor family.

1-beta-D arabinofuranosyl cytosine (Ara-C) is a highly effective drug used to treat acute leukemia. Yet, despite its high efficacy, the precise mechanism of action of ara-C mediating the cell killing and inhibition of DNA synthesis remains unclear. Ara-C is converted to the active form, ara-CTP, by cellular kinases. Ara-CTP inhibits the DNA synthesis by inhibiting DNA polymerase and blocks the elongation of the DNA strand. However, the studies using intact cells and viral system indicate that ara-C is mainly incorporated into DNA at internucleotide linkages and ara-C slows but does not terminate the DNA synthesis, suggesting the alternative mechanism of action of ara-C. We have investigated the biological effects of the incorporation of ara-Cinto DNA using the SV40 DNA replication system. Ara-C incorporation into SV40 origin caused a site specific inhibition of T-Ag (an initiator protein of DNA replication) and RP-A (human single stranded DNA binding protein) binding to the origin DNA, and reduction in the stability of DNA. Ara-C substitution at T-Ag binding site I domain inhibited more effectively the binding of T-Ag and promoted greater reduction in the stability of DNA duplexes compared to the ara-C substitution at T-Ag binding site II and early mRNA domains, and contributes to the ara-C substitutions at other sites in inhibiting T-Ag binding and reducing the stability of the DNA duplex. The ara-C substitutions at both site I and early mRNA domains also disrupted RP-A binding. Furthermore ara-C substituted origin DNA has lower affinity for T-Ag binding than unsubstituted DNA. These site specific effects of ara-C incorporation may be sequence dependent. Moreover, ara-C incorporation within the SV40 origin appears to have essentially no effect on the enzymatic unwinding of origin DNA by T-Ag helicase and the initiation and subsequent elongation of the origin DNA by the human multiprotein DNA replication complex (MRC). The data suggest that the ara-C incorporation changes the structure of DNA, which alters the binding affinity of the origin DNA for T-Ag and RP-A binding, but does not disrupt the enzymatic processes involved in replication of the DNA. Our results support the findings that: (1) ara-C is a weak competitive inhibitor of DNA polymerase; and, (2) ara-C incorporation slows, but does not terminate DNA synthesis.

Little definitive information has emerged to sufficiently describe the mechanism by which mammalian chromosomal DNA is replicated. However, it is becoming more apparent that intracellular metabolism does not occur by random collisions between soluble enzymes and substrates but rather is mediated by the action of organized multiprotein complexes. The overall objective of the research in this thesis is to characterize more fully the human cell multiprotein DNA replication complex termed synthesome with respect to its structural components, activity and evaluation of its mechanism for replication. This thesis project has presented evidence that efficient in vitro SV40 DNA replication activity by the synthesome depends on the presence of specific reaction components. DNA replication kinetics were also analyzed and a significant reduction in the lag time for initiation of replication was seen. The synthesome was evaluated for a semiconservative mechanism of DNA replication by its sensitivity to MboI digestion and isopycnic centrifugation of in vitro synthesized DNA. It was demonstrated that both DNA polymerase alpha and delta co-purify with the human cell synthesome and that antibody raised against PCNA inhibits synthesome driven in vitro DNA replication activity. Taken together these results suggest that at least two DNA polymerases are associated with the replication competent mammalian synthesome. As determined by immunoblot assays, several other proteins also exclusively co-purify with the human cell synthesome. These proteins include RP-A, topoisomerase II, RF-C, DNA ligase I and two human DNA helicases. These helicases were able to function as enzymes as demonstrated by the strand displacement assay. In addition, it was predicted that the synthesome may be tied to the mismatch repair process and it was demonstrated by immunoblot analysis that the postreplication mismatch repair gene, hMLH1, co-fractionates. These results may suggest that repair can occur during replication and that there is some benefit to coordination of progress of the replication fork and removal of lesions. Another protein thought to be involved in active DNA processes such as replication, transcription or repair is Ku. It was demonstrated that the Ku protein does co-purify along with synthesome. It was also demonstrated by immunoblot analysis that DNA polymerase varepsilon co-fractionates and thus can be included as a new component of the synthesome and that DNA polymerase beta does not co-purify with the mammalian synthesome. In addition, proteins involved with transcription and nucleotide excision repair (NER), such as RNA polymerase I, TFIIH and the excision repair XPA protein, do not co-purify with the synthesome. This suggests that transcription and NER are not an integral part of the synthesome. A model was proposed to represent this multiprotein DNA replication complex or DNA synthesome based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex. The recent data in this thesis provides additional evidence to support the premise that DNA synthesis is mediated by a multiprotein complex in mammalian cells.