Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Cleavage and activation of calpain and its substrate caspase-7 in prostate cancer cells: Evidence for a role in apoptotic sensitization

Changes in the levels of intracellular calcium mediate multiple biological effects, including apoptosis, in some tumor cells. Early studies demonstrated that prostate cancer cells are highly sensitive to alterations in the levels of their intracellular calcium pools. Furthermore, it has been established that apoptosis in prostate cancer could be initiated through calcium-selective ionophores, or inhibitors of intracellular calcium pumps. High sensitivity to changes in intracellular calcium levels may therefore be exploited as a novel mechanism for controlling prostate cancer apoptotic thresholds; however, the mechanisms associated with this process are poorly understood. To investigate the role of calcium as a mediator of prostate cancer cell death and its effects on caspase activation, LNCaP and PC-3 cell response to the calcium ionophore A23187, were examined. LNCaP cells were highly sensitive to changes in intracellular calcium, and subtoxic concentrations of A23187 facilitated apoptosis initiated by cytokines (TNF or TRAIL). In contrast, PC-3 cell death was not affected by A23187 or cytokines. A23187 caused rapid and concentration-dependent activation of calpain in LNCaP (but not PC-3 cells) which correlated with cleavage of calpain substrates caspase-7 and PTP1B. Cleavage of PTP1B from a 50 kDa to 42 kDa protein correlated with its translocation from the endoplasmic reticulum to the cytosol and with inhibition of tyrosine phosphorylation. Caspase-7 was cleaved from a 35 kDa to 30 kDa protein in response to A23187 in LNCaP (but not PC-3) cells and correlated with activation of both upstream and downstream caspases. Extracts from A23187-treated LNCaP cells, or PC-3 cells transiently transfected with calpain, mediated similar processing of in vitro transcribed and translated (TNT) caspase-7. In vitro processing of caspase-7 correlated with its proteolytic activation, which was inhibited by calpain inhibitor (calpeptin) and to some degree, by caspase inhibitors (zVAD, DEVD). Together, these results suggest that calpain is directly involved in calcium-mediated apoptosis of prostate cancer cells through activation and cleavage of caspase-7 and other substrates. Loss of calpain activation may therefore play a critical role in apoptotic resistance of some prostate cancer cells. ^

Stability and potential application of spliced nuclear pre -mRNA introns

A major portion of this thesis work was dedicated to study the nature and significance of spliced introns. The initial work was focused on studying the IVS1$\sb{\rm C\beta 1}$ intron from a T-cell receptor (TCR)-$\beta$ gene. Compared to an intron lariat control from adenovirus pre-mRNA that was spliced in vitro, IVS1$\sb{\rm C\beta 1}$ was debranched less efficiently by HeLa S100 extracts, although IVS1$\sb{\rm C\beta 1}$ also used the consensus branchpoint in vivo. Subcellular-fractionation analysis showed that most IVS1$\sb{\rm C\beta 1}$ lariats cofractionated with pre-mRNA in the nucleus, consistent with the possibility that intron degradation releases splicing factors which will be available for further rounds of splicing. The half-life of IVS1$\sb{\rm C\beta 1}$ from the endogenous TCR-$\beta$ gene was measured using the general transcription inhibitor actinomycin D to be about $\sim$15 min, which was similar to that of unstable mRNAs such as c-myc mRNA.^ The general transcription inhibitor DRB was also used for intron stability analysis. Unexpectedly, DRB decreased intron and pre-mRNA levels only initially, it later increased the levels of intron-containing RNAs. Inhibition of transcription initiation appeared to be the major early effect (the reduction phase); whereas enhanced premature transcription termination was dominant later (the induction phase).^ Having established the procedures for studying in vivo spliced introns, this approach was applied to study the mechanism of nonsense-mediated downregulation (NMD), a phenomena in which premature termination codons (PTCs) decrease the levels of mRNAs. In this study, the novel intron-oriented approach was applied to study the mechanism of NMD. The levels of spliced introns immediately upstream and downstream of a PTC-bearing exon in a TCR-$\beta$ gene were identified and analyzed along with their pre-mRNA. Although PTC reduced the mRNA levels by 4 to 9 fold, the steady-state levels of spliced introns and the pre-mRNA-to-intron ratios were not significantly altered, indicating that the PTC did not significantly inhibit TCR-$\beta$ RNA splicing. Consistent with this conclusion, the half-lives of the PTC$\sp+$ and PTC$\sp-$ pre-mRNA were similar. The protein synthesis inhibitor cyclohexmide (CHX) upregulated the levels of the PTC$\sp+$ mRNA over 10 fold without affecting the levels of the spliced introns, suggesting that the reversal effect of CHX was through stabilization, not production. These results indicated that inhibition of splicing could not be the major mechanism for the NMD pathway of the TCR-$\beta$ gene, instead, suggesting that mRNA destabilization may be more important. (Abstract shortened by UMI.) ^

Transcriptional regulation of the human {\it PAX6\/} gene

PAX6, a member of the paired-type homeobox gene family, is expressed in a partially and temporally restricted pattern in the developing central nervous system, and its mutation is responsible for human aniridia (AN) and mouse small eye (Sey). The objective of this study was to characterize the PAX6 gene regulation at the transcriptional level, and thereby gain a better understanding of the molecular basis of the dynamic expression pattern and the diversified function of the human PAX6 gene.^ Initially, we examined the transcriptional regulation of the PAX6 gene by transient transfection assays and identified multiple cis-regulatory elements that function differently in different cell lines. The transcriptional initiation site was identified by RNase protection and primer extension assays. Examination of the genomic DNA sequence indicated that the PAX6 promoter has a TATA like-box (ATATTTT) at $-$26 bp, and two CCAAT-boxes are located at positions $-$70 and $-$100 bp. A 38 bp ply (CA) sequence was located 992 bp upstream from the initiation site. Transient transfection assays in glioblastoma cells and leukemia cells indicate that a 92 bp region was required for basal level PAX6 promoter activity. Gel retardation assays showed that this 92 bp sequence can form four DNA-protein complexes which can be specifically competed by a 31-mer oligonucleotide containing a PAX6 TATA-like sequence or an adenovirus TATA box. The activation of the promoter is positively correlated with the expression of PAX6 transcripts in cells tested.^ Based on the results obtained from the in vitro transfection assays, we did further dissection assay and functional analysis in both cell-culture and transgenic mice. We found that a 5 kb upstream promoter sequence is required for the tissue specific expression in the forebrain region which is consistent with that of the endogenous PAX6 gene. A 267 bp cell-type specific repressor located within the 5 kb fragment was identified and shown to direct forebrain specific expression. The cell-type specific repressor element has been narrowed to a 30 bp region which contains a consensus E-box by in vitro transfection assays. The third regulatory element identified was contained in a 162 bp sequence (+167 to +328) which functions as a midbrain repressor, and it appeared to be required for establishing the normal expression pattern of the PAX6 gene. Finally, a highly conserved 216 bp sequence identified in intron 4 exhibited as a spinal cord specific enhancer. And this 216 bp cis-regulatory element can be used as a marker to trace the differentiation and migration of progenitor cells in the developing spinal cord. These studies show that the concerted action of multiple cis-acting regulatory elements located upstream and downstream of the transcription initiation site determines the tissue specific expression of PAX6 gene. ^

The effect of DNA methylation in carcinogenesis

CpG island methylation within single gene promoters can silence expression of associated genes. We first extended these studies to bidirectional gene pairs controlled by single promoters. We showed that hypermethylation of bidirectional promoter-associated CpG island silences gene pairs (WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF 195580) simultaneously. Hypomethylation of these promoters by 5-aza-2'-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. These results were further confirmed by luciferase assays. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemia, respectively. ^ Next we sought to understand the origins of hypermethylation in cancer. CpG islands associated with tumor suppressor genes are normally free from methylation, but can be hypermethylated in cancer. It remains poorly understood how these genes are protected from methylation in normal tissues. In our studies, we aimed to determine if cis-acting elements in these genes are responsible for this protection, using the tumor suppressor gene p16 as a model. We found that Alu repeats located both upstream and downstream of the p16 promoter become hypermethylated with age. In colon cancer samples, the methylation level is particularly high, and the promoter can also be affected. Therefore, the protection in the promoter against methylation spreading could fail during tumorigenesis. This methylation pattern in p16 was also observed in cell lines of different tissue origins, and their methylation levels were found to be inversely correlated with that of active histone modification markers (H3K4-3me and H3K9-Ac). To identify the mechanism of protection against methylation spreading, we constructed serial deletions of the p16 protected region and used silencing of a neomycin reporter gene to evaluate the protective effects of these fragments. A 126 bp element was identified within the region which exerts bidirectional protection against DNA methylation, independently of its transcriptional activity. The protective strength of this element is comparable to that of the HS4 insulator. During long-term culture, the presence of this element significantly slowed methylation spreading. In conclusion, we have found that an element located in the p16 promoter is responsible for protection against DNA methylation spreading in normal tissues. The failure of protective cis-elements may be a general feature of tumor-suppressor gene silencing during tumorigenesis. ^

Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma.

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.

Phenotypic and genotypic analysis of mouse hepatitis virus (JHM) complementation group A temperature-sensitive mutants

Temperature sensitive (ts) mutant viruses have helped elucidate replication processes in many viral systems. Several panels of replication-defective ts mutants in which viral RNA synthesis is abolished at the nonpermissive temperature (RNA$\sp{-})$ have been isolated for Mouse Hepatitis Virus, MHV (Robb et al., 1979; Koolen et al., 1983; Martin et al., 1988; Schaad et al., 1990). However, no one had investigated genetic or phenotypic relationships between these different mutant panels. In order to determine how the panel of MHV-JHM RNA$\sp{-}$ ts mutants (Robb et al., 1979) were genetically related to other described MHV RNA$\sp{-}$ ts mutants, the MHV-JHM mutants were tested for complementation with representatives from two different sets of MHV-A59 ts mutants (Koolen et al., 1983; Schaad et al., 1990). The three ts mutant panels together were found to comprise eight genetically distinct complementation groups. Of these eight complementation groups, three complementation classes are unique to their particular mutant panel; genetically equivalent mutants were not observed within the other two mutant panels. Two complementation groups were common to all three mutant panels. The three remaining complementation groups overlapped two of the three mutant sets. Mutants MHV-JHM tsA204 and MHV-A59 ts261 were shown to be within one of these overlapping complementation groups. The phenotype of the MHV-JHM mutants within this complementation class has been previously characterized (Leibowitz et al., 1982; Leibowitz et al, 1990). When these mutants were grown at the permissive temperature, then shifted up to the nonpermissive temperature at the start of RNA synthesis, genome-length RNA and leader RNA fragments accumulated, but no subgenomic mRNA was synthesized. MHV-A59 ts261 produced leader RNA fragments identical to those observed with MHV-JHM tsA204. Thus, these two MHV RNA$\sp{-}$ ts mutants that were genetically equivalent by complementation testing were phenotypically similar as well. Recombination frequencies obtained from crosses of MHV-A59 ts261 with several of the gene 1 MHV-A59 mutants indicated that the causal mutation(s) of MHV-A59 ts261 was located near the overlapping junction of ORF1a and ORF1b, in the 3$\sp\prime$ end of ORF1a, or the 5$\sp\prime$ end of ORF1b. Sequence analysis of this junction and 1400 nucleotides into the 5$\sp\prime$ end of ORF1b of MHV-A59 ts261 revealed one nucleotide change from the wildtype MHV-A59. This substitution at nucleotide 13,598 (A to G) was a silent mutation in the ORF1a reading frame, but resulted in an amino acid change in ORF1b gene product (I to V). This amino acid change would be expressed only in the readthrough translation product produced upon successful ribosome frameshifting. A revertant of MHV-A59 ts261 (R2) also retained this guanidine residue, but had a second substitution at nucleotide 14,475 in ORF1b. This mutation results in the substitution of valine for an isoleucine.^ The data presented here suggest that the mutation in MHV-A59 ts261 (nucleotide 13,598) would be responsible for the MHV-JHM complementation group A phenotype. A second-site reversion at nucleotide 14,475 may correct this defect in the revertant. Sequencing of gene 1 immediately upstream of nucleotide 13,296 and downstream of nucleotide 15,010 must be conducted to test this hypothesis. ^

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.

Calcium mobilization from the endoplasmic reticulum to the mitochondria during apoptosis in prostate cancer

Previous studies have implicated Ca2+ fluxes in the control of apoptosis but their exact roles in regulating the process remain obscure. Because Ca2+ can serve as a signal for cytochrome c release from isolated mitochondria, we hypothesized that alterations in intracellular Ca2+ compartmentalization might serve as a release signal in whole cells undergoing apoptosis. Exposure of human PC-3 prostate adenocarcinoma cells to staurosporine or DNA damaging agent (doxorubicin) but not to anti-Fas antibody led to early release of Ca2+ from the endoplasmic reticulum and subsequent accumulation of Ca2+ within mitochondria. Both events were blocked in cells stably transfected with Bcl-2 but were not affected by treatment with the pancaspase inhibitor, zVADfmk. The effects of staurosporine were associated with re-localization of Bax from the cytosol to both endoplasmic reticular and mitochondrial membranes. Neither ER Ca 2+ pool depletion nor mitochondrial Ca2+ uptake were observed in DU-145 cells that possess a frameshift mutation in the Bax gene unless wild-type Bax was restored via adenoviral gene transfer. Cytochrome c release and downstream features of apoptosis were attenuated by treatment with an inhibitor of mitochondria) Ca2+ uptake (RU-360). Although, direct pharmacological ER Ca2+ pool emptying in cells treated with thapsigargin did not lead to early cytochrome c release, pretreatment of cells with staurosporine dramatically sensitized mitochondria to thapsigargin-induced cytochrome c release. Together, our data demonstrate that ER-to-mitochondrial Ca2+ fluxes promote cytochrome c release and apoptosis in cells exposed to some (but not all) pro-apoptosic stimuli. ^

Analysis of Wnt/beta-catenin signaling in kidney tubulogenesis

The β-catenin/Lef/Tcf-mediated Wnt pathway is central to the developmental of all animals, stem cell renewal, and cancer progression. Prior studies in frogs and mice have indicated that the ligand Wnt-4 is essential for the mesenchyme to epithelial transition that generates tubules in the context of kidney organogenesis. More recently, Wnt-9b in mice, was likewise found to be required. Yet despite the importance of Wnt signals in renal development, the corresponding Frizzled receptor(s) and downstream signaling mechanim(s) are unclear. My work addresses these knowledge gaps using in vitro (Madin-Darby Canine Kidney cells) and in vivo (Xenopus laevis and zebrafish pronephros) tubulogenic kidney model systems. Employing established reporter constructs of Wnt/β-catenin pathway activity, I have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions. I have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/β-catenin pathway using β-Engrailed and dnTCF-4, constructs that suppress this pathway. I have further found that MDCK cells express the Frizzled-6 receptor, and that Wnt-4 forms a biochemical complex with Frizzled-6, yet does not appear to transduce Wnt-4's canonical signal. Additionally, I demonstrate that standard Hepatocyte Growth Factor (HGF)-mediated (non-physiologic) induction of MDCK tubulogenesis in collagen matrices is not altered by activation or suppression of β-catenin signaling activity; however, β-catenin signaling maintains cell survival in this in vitro system. Using a Wnt/β-catenin signaling reporter in Xenopus laevis, I detect β-catenin signaling activity in the early pronephric epithelial kidney tubules. By inhibiting the Wnt/β-catenin signaling pathway in both zebrafish and Xenopus , a significant loss of kidney tubulogenesis is observed with little or no effect on adjoining axis or somite development. This inhibition also leads to the appearance of severe edema that phenocopies embryos depleted for Wnt-4. Tubulogenic loss does not appear to be caused by increased cell death in the Xenopus pronephric field, but rather by lessened expression of tubule epithelium genes associated with cellular differentiation. Together, my results show that Wnt/β-catenin signaling is required for renal tubule development and that Wnt-4 is a strong candidate for activating this pathway. ^