Thromboxane synthase (TS) catalyzes the conversion of prostaglandin H{dollar}\sb2{dollar} into thromboxane A{dollar}\sb2{dollar} (TxA{dollar}\sb2),{dollar} which is a potent mediator of platelet aggregation and vasoconstriction. A deficiency of platelet TS or mutations in the TxA{dollar}\sb2{dollar} receptor gene have been shown to result in bleeding disorders, while an elevated level of TxA{dollar}\sb2{dollar} may be associated with cardiovascular and renal diseases. Several post-transcriptional events have been demonstrated to curtail the level of TS in vivo, presumably for preventing over-production of the autacoid. At present, little is known about the transcriptional regulation of TS gene expression. To address this, a genomic DNA containing the human TS promoter was cloned and characterized. 5{dollar}\sp\prime{dollar}-RACE (rapid amplification of cDNA ends) and ribonuclease (RNase) A/T1 protection assays revealed multiple transcription initiation sites. Deletion analysis indicated that TS transcription is mainly TATA-independent. A proximal positive regulatory sequence (PPRS, {dollar}-{dollar}90 to {dollar}-{dollar}25 bp) and several distal repressive elements, including a silencer, were also identified in the promoter. The PPRS worked in an orientation-independent but position-dependent manner, and could be further divided into two independent elements, PPRS{dollar}\sb1{dollar} ({dollar}-{dollar}90 to {dollar}-{dollar}50 bp) and PPRS{dollar}\sb2{dollar} ({dollar}-{dollar}50 to {dollar}-{dollar}25 bp). While similar amounts of nuclear factor(s) from different cell types may interact with PPRS{dollar}\sb2,{dollar} those interacting with PPRS{dollar}\sb1{dollar} exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS{dollar}\sb1{dollar} ({dollar}-{dollar}60 tgctgattcat {dollar}-{dollar}50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by RT-PCR amplification of the cDNA and Northern analysis. A 9-fold trans-activation of luciferase (luc) reporter gene expression was detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite that NF-E2 and the cis-elements could alter the level of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status of the TS promoter in several human cell lines revealed cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.

The emergence of drug-resistance in cancer cells during chemotherapy remains a major obstacle in the treatment of neoplasia. Multidrug resistance (MDR) to a group of unrelated cytotoxic compounds can be conferred to eucaryotic cells by the expression of P-glycoprotein (Pgp), a putative plasma membrane transporter believed to mediate the efflux of these agents out of cells. A variety of agents are able to reverse this MDR phenotype by inhibiting the Pgp transporter. Blocking the action of this protein increases the effectiveness of cancer chemotherapeutic agents and, hence, has significant clinical implications. A mutant Pgp1 cDNA containing the substitution (Gly{dollar}\sp{lcub}338{rcub}{dollar}Ala{dollar}\sp{lcub}339{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}Pro{dollar}\sp{lcub}339{rcub}{dollar}) within the sixth transmembrane domain (tm6) has been cloned. The expression of this mutant confers an MDR phenotype preferentially resistant to actinomycin D. In this thesis we report that this MDR phenotype also has a decreased sensitivity toward reversal by cyclosporin A, while the sensitivity toward verapamil is unaltered. The accumulation of {dollar}\rm\lbrack\sp3H\rbrack{dollar} vincristine in cells expressing the wild-type Pgp1, not the mutant, increases dramatically in the presence of cyclosporin A, which correlates well with the reversal profile. We have altered only one amino acid residue at this location (Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar} or Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar}). The transfectants expressing the Pgp1 containing the proline substitution, rather than the alanine, demonstrate an MDR phenotype which is preferentially resistant to actinomycin D, and insensitive to reversal by cyclosporin A. Modeling the whole tm6 domain (with the Quanta modeling program and energy minimization by the CHARMm program) reveals that the proline substitution at position 339 rather than the alanine at 338 drastically changes the local {dollar}\alpha{dollar}-helice conformation, especially the polar side chain alignment along the hydrophilic side of this amphipathic {dollar}\alpha{dollar}-helice. We hypothesize that the Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar} substitution, rather than the Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}, is the primary contributor to the aforementioned altered phenotype. We have also attempted to determine the functions of two spliced variants of Pgp1, previously cloned in this laboratory, by expressing them in an in vitro system. The biogenesis of one of the variants, ADX124, has also been investigated. We conclude that it is derived from a splicing event that involves the internal splicing signals that are maintained in the mature full-length Pgp1 transcript.

Robertsonian translocation is the result of short arm fusion between acrocentric chromosomes. It is the most common structural chromosome aberration in humans. The majority of Robertsonian translocations are dicentric and segregate normally through meiosis and mitosis. Therefore, it is assumed that one centromere is inactivated or suppressed to ensure proper transmission of the translocation chromosome in cell divisions. A single study has specifically investigated the active and inactive centromeres of only two types of Robertsonian translocations, a task which has been complicated by the lack of criteria for either an active or inactive centromere. In this study, several approaches were applied in the identification of the active and inactive centromeres of dicentric Robertsonian translocations. Dual color fluorescence in situ hybridization (FISH) of {dollar}\alpha{dollar}-satellite DNA probes was used to assess centromeric activity based on DNA morphology in 54 dicentric, nonhomologous Robertsonian translocations. By in situ analysis, 90% (49/54) of the translocations demonstrated preferential activity of a particular centromere. The chromosome 14 centromere was most often active in its translocations, while the chromosome 15 centromere was least often active when involved in Robertsonian translocation. While the DNA components of active versus inactive centromeres have not been conclusively defined, essential centromere associated proteins (CENPs) have been localized in and around the primary constriction. Two of these centromeric antigens, CENPs-C and E, are thought to bind active centromeres only; thus application of antibodies to these two antigens is a more accepted and definitive means of differentiating between active and inactive centromeres of Robertsonian translocations. Nine of ten different translocations studied bound CENPs-C and E at one centromere only, confirming the active centromere as assigned by DNA in situ hybridization. The remaining translocation, interpreted as truly dicentric based on two constricted fluorescent hybridization signals in most cells, demonstrated CENP-C and CENP-E at both centromeres. To investigate if chromosome structure is similar among Robertsonian translocations, the residual short arm content of each translocation was studied with DNA probes for the acrocentric short arm region. The most distal repetitive DNA, {dollar}\beta{dollar}-satellite, was not retained in any translocation. However, most translocation chromosomes retained satellite III, a short arm, repetitive DNA located adjacent to the {dollar}\alpha{dollar}-satellite DNA of the primary constriction. All of the t(13q14q)s which were analyzed retained a proximal satellite III subfamily, pTRS-47 only, thus demonstrating grossly identical pericentromeric structure. The t(14q21q)s, however, varied in short arm DNA composition in that no, one or two satellite III DNA subfamilies were present on the translocation chromosomes. This study is the first to demonstrate preferential centromeric activity in almost every type of Robertsonian translocation and provides evidence for a functional centromeric hierarchy. Furthermore, this is the only study to show selective binding of centromere-associated proteins to Robertsonian translocations. The results additionally indicate that translocation structure can very depending on the chromosomes involved and may influence the activity of certain acrocentric centromeres.

Cyclins and their associated catalytic subunits, cyclin-dependent kinases (CDKs), are the major regulators for the advance of cells through their growth cycle. Those cyclins that function in the G1 phase of the cell cycle play an especially important role in cellular growth regulation, as the major decision governing proliferation, quiescence and differentiation is made during this period. In addition to their roles as cell cycle regulators in the G1 phase, the three D-type cyclins (cyclin D1, D2, and D3) are intimately involved in differentiation, transcriptional activation, and signal transaction pathway. For examples, D2 and D3, but not D1, regulate G-CSF-induced myeloid differentiation; D1 physically interacts with the estrogen receptor and activates transcription in the absence of the ligand; and D2 might be a down stream mediator for the FSH-dependent signaling pathway. The structural basis that determines the differential activities of D-type cyclins is not clear. To study the roles of cyclin D2 in differentiation, transcription, and signaling, several cyclin D2 overexpressing cell lines were developed using retrovirus-mediated gene transfer. The characterization of these cell lines revealed that overexpression of cyclin D2 shortens the G1 but prolongs the S phase, alters the expression of several genes in the G1 phase of the cell cycle, and inhibits hemin-induced differentiation of K562 cells toward the erythroid lineage. Most interestingly, cells overexpressing cyclin D2 exhibited enhanced transcription capacity for different viral or cellular promoters. The increase in transcription capacity could be reproduced by transient transfection of four different types of cells with an expression vector containing the cyclin D2 cDNA. The effect was cyclin D2-specific, as other cyclins, CDKs and several transcriptional activators had no or significantly less effect. In an attempt to localize critical motifs of cyclin D2 for transcriptional activation, several cyclin D2 mutants were constructed. It was found that the mutations at RB- or CDK-binding site did not adversely affect cyclin D2's ability to enhance reporter transcription and that the critical sequence for transcriptional activation lied in the C-terminal region of cyclin D2. In addition, cyclin D2 overexpression also resulted in the activation of a novel nuclear kinase. These findings provide a potential mechanism linking cell cycle progression to transcriptional activation and signal transaction.

Src-family protein tyrosine kinases (PTKs) are critical components of T and B lymphocyte signaling cascades. To examine the relationship between Src-PTK expression and lymphocyte development, we defined quantitative and qualitative expression patterns of Src-PTKs during lymphocyte ontogeny. A quantitative RT-PCR assay was designed to distinguish transcripts derived from the lck proximal (Type I) vs distal (Type II) promoter elements, and fynT vs fynB isoforms. These assays were then used to measure lck and fyn expression in normal fetal and adult tissue. Changes in Type I and Type II promoter utilization correlate with pre-TCR and alphabetaTCR expression suggesting that as receptor complexes develop they couple to different intracellular signaling cascades that differentially regulate lck expression. The fynT isoform, not fynB, is detected in both lineages, including pre-T and pre-B cells, suggesting a role for Fyn in lymphocyte development before TCRalphabeta or IgM expression. In spite of much lower levels of fynT and lck transcripts in the B cell lineage, compared to lck transcripts in thymocytes; accumulation of lck and fynT correlates temporally with acquisition of immune receptor expression in both lineages. In addition to immune receptors, Src family PTKs are activated by receptors regulating growth and differentiation in thymic stroma which is required for lymphocyte development. To examine the impact of Src-PTK function in thymic stroma on lymphocyte development, a transgenic model system was developed in which the expression of polyoma virus middle T antigen (PymT), a molecule that activates Src-PTKs, was directed to thymic subcapsular epithelial cells using a mutant Class II promoter element. PymT expression constitutively activated endogenous Src-PTKs which phosphorylated PymT, inducing a PymT association with p85, the regulatory subunit of PI 3-kinase. Src-PTK activation was associated with changes in tyrosine phosphorylation of cellular proteins. Transgene expression in subcapsular stroma resulted in upregulation of epidermal growth factor (EGF) receptor and 4 to 5-fold thymic lymphoid hyperplasia. However, normal thymic architecture, proliferation and apoptotic responses, thymocyte differentiation, repertoire selection, and thymic involution were maintained. These findings suggest that the level and/or persistence of signals generated via Src-PTKs in thymic subcapsular epithelium serve as major defining factors influencing homeostatic control of thymic organ size and cellularity.

Germline mutations of the breast cancer associated gene 1 (BRCA1 ) predispose women to breast and ovarian cancers. In mice, loss of function mutations of Brca1 results in recessive lethality during gestation. Analysis of these mutant embryos revealed that Brca1 plays essential roles in maintaining genomic integrity. However the mechanisms underlying these defects remain to be illustrated. To further define the processes through which BRCA1 maintains genomic integrity, meiosis in mutant mice that are homozygous for a targeted deletion of Brca1 exon 11 (Brca1Delta11/Delta11) was studied. Brca1Delta11/Delta11 embryos died at early gestation, but survived to adulthood if either one or both wild type p53 alleles were also mutated. This study showed that all Brca1Delta11/Delta11 p53+/- and Brca1 Delta11/Delta11p53-/- males were infertile and females exhibited decreased fertility. Mutant males had significantly reduced testicular size and contain no spermatozoa even though mutant testes contained spermatogonia, stem cells, and early stage primary spermatocytes; in their seminiferous tubes. Chromosomal analyses on the spermatocytes indicated that mutant homologous chromosomes paired and advanced to the pachytene stage, however mutant mice were unable to form chiasma, and no spermatocytes developed to the diplotene stage. The block of spermatogenesis occurred right after homologous chromosome pairing at meiosis I. Brca1-Delta11 mutation decreased the frequency of Mlh1 foci formation at crossover sites, affected Rad51 foci formation, and interfered with the relocalization of gamma-H2AX from sites of DNA double strand breaks to the XY body during spermatogenesis. The expression levels of a number of genes that are involved in DNA damage repair were reduced including helicase and silent information regulatory proteins. Termination of spermatogenesis at pachytene stage was accompanied by increased rates of apoptosis. cDNA microarray analyses demonstrated changes in expression of genes involved in both p53-dependent and -independent apoptotic pathways. In conclusion, this study revealed an essential role for Brca1 in meiotic recombination repair and regulation of genetic exchange of homologous chromosomes during spermatogenesis. Loss of Brca1 function led to the accumulation of DNA damage, genomic instability, activation of both p53-dependent and -independent apoptotic pathways and, eventually, failure of spermatogenesis.

Two polymorphic alleles encoding dihydrofolate reductase (DHFR), exist in Chinese hamster lung (CHL) cells. One allele codes for a 21 KD enzyme, while the other codes for a 20 KD enzyme. Upon selection in methotrexate (MTX), either allele, but not both, may be amplified, resulting in a MTX-resistant phenotype. Three major DHFR transcripts 1000, 1650 and 2150 nt in length are expressed from each allele. The expression of these mRNAs is allele-specific, i.e. the 2150 nt mRNA is the most abundant species derived from the 21 K allele, while the 1000 nt mRNA is the most abundant species derived from the 20 K allele. DC- 3F/MQ8, a cell line overexpressing the 20 K allele, has a unique RNA expression profile. In this cell line, both the 1000 nt and the 1650 nt mRNA species are overexpressed to the same extent in contrast to what is seen with other 20 K-overexpressors in which only the 1000 nt species is predominantly overexpressed. Previous studies suggested that the allele specific DHFR RNA expression in CHL cells might be related to differential utilization of the three poly(A) sites present in each allele. As a first step, to study the unique RNA expression profile in DC- 3F/MQ8, we have cloned and sequenced a 2.1 kb BamH1 fragment extracted from DC-3F/MQ8 cells that contained the three major DHFR poly(A) sites. In comparing this fragment with the same fragment from another 20 K-overexpressing cells, DC-3F/MQ19, we found no sequence differences. Thus, the unique RNA expression profile in DC-3F/MQ8 could not be related to sequence changes in the 3{dollar}\sp\prime{dollar}UTRs of the DHFR mRNAs. Using PCR-mediated RNase protection assays, we have mapped the 3{dollar}\sp\prime{dollar} ends of the three major DHFR mRNAs from each allele. While the first two poly(A) sites were mapped to the same positions in the two alleles, the third poly(A) site was located at the different positions between the two alleles. The 2.1 kb BamH1 fragment containing the three poly(A) sites from each allele was then cloned into a SV40 based transfection vector downstream of a CAT reporter gene, and the vectors thus constructed were transiently expressed in COS cells. As a result, an allele-specific CAT RNA expression profile was reproduced. A four-base deletion has been found at the 3{dollar}\sp\prime{dollar} end of the third poly(A) signal sequence in the 20 K allele so that the consensus sequence AAUAAA is altered to AAUAAU in this allele. Site-directed mutagenesis studies have demonstrated that this four-base deletion is primarily responsible for the dramatic alterations in 3{dollar}\sp\prime{dollar} polyadenylation between the two alleles. Finally, our studies indicated that in spite of the pronounced differences in 3{dollar}\sp\prime{dollar} polyadenylation of DHFR mRNA between the two alleles, DHFR gene expression between the two alleles was essentially unchanged. Thus, the allelic differences in 3{dollar}\sp\prime{dollar} processing of DHFR mRNAs can not be the basis for the three-fold selective preference of the 21 K allele over the 20 K allele observed in CHL cells during long term MTX selection.

In transformed cells the processing and translocation of lysosomal proteases are altered and protease inhibitors are deregulated. These alterations increase the capacity of neoplastic cells for degradation of extracellular matrix constituents and are thought to contribute to tumor invasion. I have tested the hypothesis that deregulation of intracellular protein degradation by lysosomes may also contribute to tumorigenesis by conferring a growth advantage to neoplastic cells, particularly in conditions of nutritional stress. In these conditions normal cells up-regulate the degradation of their cytoplasmic proteins to maintain amino acid and glucose homeostasis. To this end, I have studied protein turnover and the inducibility of lysosomal protein degradation in one mesenchymal (F9 12-1a) and three bronchial epithelial (BEAS-2B, a SV40 transformed clone of normal human bronchial cells (NHBE); BZR, a ras transfected tumorigenic clone of BEAS-2B; Calu-1, an epidermoid carcinoma cell line) transformed cells in culture. Protein degradation and synthesis were measured as the release of {dollar}\rm\sp{lcub}14{rcub}C{dollar}-valine from cells and incorporation of {dollar}\rm\sp{lcub}14{rcub}C{dollar}-valine into proteins. Autophagic degradation of cytoplasm was quantified by ultrastructural morphometry. I found that in F9 12-1a, BEAS-2B, and BZR cells basal proteolysis was down-regulated. In the transformed bronchial cells basal protein degradation was unaffected by cell density and did not appear to be regulated in parallel with rates of protein synthesis and cell proliferation. Prolonged nutritional deprivation up-regulated basal proteolysis in Calu-1 cells, this change was associated with the development of a more differentiated cellular phenotype. The induction of malignant phenotype did not affect basal proteolysis. Amino acid deprivation stimulated autophagic protein degradation equally well (2-fold) in transformed and normal cells. Serum growth factors did not regulate proteolysis in either normal or transformed bronchial cells. Induction of differentiated phenotype by {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar} or prolonged nutritional deprivation increased the inducibility of autophagic protein degradation in normal and transformed epithelial cells and upregulated proteolysis in response to deprivation of growth factors in Calu-1 cells. I conclude that transformation decreases the basal degradation of cytoplasmic proteins in some neoplastic cells. Down-regulation of proteolysis occurs independently of protein synthesis. Lysosomal proteolysis of transformed cells is not down-regulated. In normal and transformed cells proteolysis can be strikingly augmented by autophagy during acute or chronic nutritional stresses and can be further enhanced by inducing cell differentiation.

Xeroderma pigmentosum (XP) has been a focus of extensive investigation since its clinical description in 1874 and the subsequent demonstration of a DNA repair defect in 1968. However, identification of the mutant genes responsible for XP has been hindered by numerous experimental limitations and extensive genetic heterogeneity. The obvious in vitro phenotypes of UV sensitivity and defective DNA repair observed in XP fibroblasts can be complemented following transfer of chromosomes from normal cells. This strategy offers an excellent approach for gene mapping and cloning of the defective genes. Recently, a rearranged single human chromosome (Tneo) has been isolated which restores both UV light resistance and DNA repair activity in XP complementation group D (XP-D) cells. The cytogenetic and molecular characterization of Tneo was the focus of this dissertation. The results provided preliminary mapping of the XP-D locus and serve as a critical first step in the isolation of the gene(s) defective in XP-D cells. Classical cytogenetic analysis revealed that Tneo is a monocentric chromosome of undefined human origin. "Reverse in situ hybridization" by Alu-PCR indicated that this chromosome involves a complex rearrangement consisting of chromosome segments from 16q, 17p and 19. "Painting" with chromosome-specific DNA libraries was used to define the orientation of the chromosomal segments present in Tneo. Southern blot hybridizations using chromosome-specific probes further delineated the specific breakpoints involved in the Tneo rearrangement, as consistent with a der(16)(19pter{dollar}\to{dollar}19p13.2::17p?{dollar}\to{dollar}17p?::19p13.1{dollar}\to{dollar}19p13.1::16cen{dollar}\to{dollar}16q23q24?:: 19q13.2{dollar}\to{dollar}19q13.3::17p?{dollar}\to{dollar}17p?). Deletions of the complementing chromosome were generated and revealed that the restoration of UV resistance in XP-D cells resides in chromosomal material in the distal q-arm of Tneo. Molecular characterization demonstrated that the XP-D complementing region involves DNA derived from 19q13.2{dollar}\to{dollar}19q13.3 and 17p. Transfer of a normal human chromosome 17 into XP-D cells failed to correct the defective phenotypes, implying that the XP-D gene lies within or near 19q13.2{dollar}\to{dollar}19q13. It is of particular interest that this region includes a previously described human DNA repair gene cluster. The experiments described have generated materials which should prove valuable for the molecular cloning of the XP-D gene(s). Additionally, the data illustrate the power of combined cytogenetic and molecular mapping techniques for use in the isolation of genes defective in a variety of human genetic disorders which exhibit in vitro phenotypes.