Physiological concentrations of the pineal hormone melatonin inhibit the proliferation of estrogen-dependent (MCF-7), but not estrogen-independent (MDA-MB-330) breast cancer cells. Although both cell lines express low levels of the G protein-coupled mt1 melatonin receptor, higher levels of receptor are expressed by MCF-7 cells. To determine if the mt1 receptor mediates melatonin's growth-suppressive effect we examined several parameters m MCF-7 and MDA-MB-330 breast cancer cells stably-transfected with, and overexpressing the mt1 receptor (mt1-MCF-7, mt1-MDA). Using these transfected cells, a 46% enhancement of melatonin's growth-suppressive effects was seen in mt1-MCF-7 cells, however, MDA-MB-330 cells remained insensitive to melatonin's growth-inhibition. The mtl receptor agonist and antagonist demonstrated that these growth-inhibitory effects of melatonin in MCF-7 cells were mtl specific. As well, mt1 overexpression elicited MCF-7 cells supersensitive to melatonin's gene-modulatory effects. Melatonin enhanced pS2 expression in mt1-overexpressing cells by 145%, and significantly inhibited expression of ERalpha We then examined coupling of the mt1 receptor in parental, and mt1-overexpressing MCF-7 cells. Pertussis toxin was used so determine if inhibition of G alphai function would lead to a block of melatonin's growth-inhibitory effects However, melatonin's growth-inhibitory effects were determined to be insensitive to pertussis toxin in both parental and mt1-transfected MCF-7 cells. Yet, melatonin did inhibit forskolin-induced cAMP accumulation in parental and mtl transfected MCF-7 cells. Taken together, these data suggest that although the Galphai proteins do not appear to be the mediators of melatonin's growth-inhibitory effects, melatonin inhibits cAMP accumulation, suggesting that multiple pathways are involved In nude mouse tumor studies using mtl-transfected MCF-7 cells, mt1-overexpression significantly decreased tumor burden and weight. This inhibition was enhanced by melatonin Taken together, these data demonstrate that the mt1 receptor mediates melatonin's growth-inhibitory effects in ERalpha-positive human breast cancer cells, and that overexpression of the receptor results in a melatonin supersensitive phenotype. However, this effect depends upon expression of ERalpha. These data also demonstrate that the mt1 receptor mediates melatonin's growth-inhibitory effects in vivo and overexpression of the receptor dramatically inhibits proliferation and sensitizes tumors to melatonin's growth-inhibitory effects / acase@tulane.edu

Bone marrow toxicity induced by specific anticancer agents and/or ionizing radiation remains a major limitation in clinical management of cancer. Metallothionein proteins (MTs) scavenge free radicals and can act as nucleophilic proteins to react with electrophilic anticancer drugs. U-937 and K-562 myeloid progenitor cell lines were transfected with a murine retroviral vector encoding the human MT-IIa gene (pLMTSN), pLtripMTSN (encoding an additional enhancer in addition to hMT-IIa) or the control pLXSN using viral supernatants or cocultivation in the presence of an intracellular protein peptide carrier polybrene (8 mug/ml). Upregulated MT levels in the transfected cells were demonstrated by RT-PCR analysis, immunocytochemistry, differential silver staining after SDS-PAGE, and 109Cd/hernoglobin affinity assays. Azacytidine (azaC, 4 muM), an inhibitor of DNA methylation, further enhanced MT expression in K-562 cells transfected with MT gene. Transfection of MT gene in U-937 cells caused a 2.1 fold increase in MT levels. Exposure of these cells to Zn2+ (100 muM), increased the MT levels by 26.8 fold as measured by 109Cd/hemoglobin affinity assays. Transfected U-937 cells exposed to 100 cGy, cisplatin (30 ng/ml), or melphalan 0.3 mug/ml) led to significant increases in cell survival by 6, 2.8 and 1.9 fold, respectively, as monitored by the colony forming assay. Pretreating transfected U-937 cells with Zn2+ (40 muM) resulted in a 1.6--2.2 fold increase in cell survival after 14--17.5 Gy exposure as monitored by the MTT assay. Antioxidant potential was significantly increased in K-562 and U-937 cells transfected with MT as monitored by chemiluminescence assay. Significant protection from radiation induced apoptosis was observed in MT transfected U-937 cells pretreated with Zn2+ (40 muM) and exposed to 20 Gy (post 6h). Anti-apoptotic bcl-Xl protein levels were found to be increased in MT expressing cells which was further accentuated by Zn2+, while bcl-2 and the proapoptotic bax were found to decrease as monitored by Western blots and RT-PCR. These results indicate that transfection of the bone marrow cells with the MT gene may provide an important therapeutic approach for their protection by imparting a resistance phenotype that acts by scavenging free radicals and modulating the expression of specific proteins to inhibit radiation or drug induced apoptosis in a p53 independent pathway / acase@tulane.edu

In this dissertation, test statistics for determining whether the k $(k\geq2)$ Weibull location parameters are equal are given both for censored and complete samples. For the case when the shape parameter, $\beta,$ of the Weibull distribution is equal to unity, the exponential case, simpler test statistics are provided that are functions of the means and first order statistics The asymptotic distribution of the test statistic is derived for two complete samples from exponential populations. The power of the test of equality of two location parameters for complete samples from exponential populations is derived using a new approach. Ten thousand exponential samples, with the desired mean and location parameter are simulated using the International Mathematical and Statistical Libraries software (Visual Numerics, Inc., Houston, 1991) and the number of times the null hypothesis (of equality of two location parameters) is rejected is then recorded. Simulated power is the proportion of the samples for which the null hypothesis is rejected Generally, there is good agreement between calculated and simulated power. Power depends on the ratio of hazards, sample size, the magnitude of the difference between location parameters and the level of significance. Fixing any three of the preceding parameters and increasing the fourth results in increased power / acase@tulane.edu

The recombinant retrovirus, MoFe2, was constructed by replacing the U3 region of Moloney murine leukemia virus (MoMuLV) with homologous sequences from the feline leukemia virus (FeLV-945) LTR. Like other gammaretroviruses, MoMuLV and FeLV induce lymphoma in the natural host through insertional activation of host oncogenes near proviral integrations sites. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. Southern blot analysis demonstrated that the MoFe2 recombinant did not integrate near the host oncogenes commonly involved in disease induction by either parent virus; thus, MoFe2 represents an opportunity to identify novel oncogenes in the induction of T-cell lymphoma. Common insertion sites (CISs) in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parental virus in wild type animals. Two of the newly identified CISs, 3-19 and Rw1, had not been previously implicated in lymphoma in any retroviral model. The exact function of Rw1 remains unknown and little information is available about the regulation of its expression. The precise location and transcriptional direction of proviral insertions in Rw1 were determined, but proviral insertion demonstrates no clear impact on the level of Rw1 expression in end-stage lymphomas. The results further indicate that Rw1 expression is highly regulated, restricted to a narrow spectrum of cell types, and may be responsive to retroviral infection. The other novel CIS, designated 3-19, encodes the regulatory subunit of phosphatidylinositol 3-kinase gamma (P13Kgamma) termed p101. P13Kgamma is known to be important in a number of cellular functions including chemotaxis, oxidative burst and regulation of T-cell proliferation. P13Kgamma activity is regulated through interactions with G protein-coupled receptors. The fact that p101 is a CIS suggests that its dysregulated expression is associated with T-cell malignancy. Tumors that contained insertions in 3-19 had elevated expression of one or more of the detected transcripts. Overexpression studies revealed that p101 has a positive effect on PI3K by activating Akt, likely by enhancing the activity of p110gamma and sensitizing it to activation. Although overexpression of p101 activates the Akt pathway and does protect cells from DNA damage-induced apoptosis it was also found to induce apoptosis. These findings indicate that substitution of FeLV-945 U3 sequences into the M-MuLV LTR significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma and supports a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. These findings demonstrate the utility of the recombinant retrovirus MoFe2-MuLV to in the identification of novel genes implicated in the induction of lymphoma / acase@tulane.edu

SIV infection in the rhesus macaque has been intensively studied as a model of AIDS-related non-Hodgkin's lymphoma (ARL). Previous studies show that SAIDS-related non-Hodgkin's lymphoma (sARL) in the rhesus macaque recapitulates the major pathobiologic features of AIDS-NHL. However, beyond infection with RhLCV, little is known about what might be driving the growth and survival of sARL cells. This study investigates the regulation of growth and apoptosis in sARL cells to determine how sARL models human ARL. First, a homolog of KSHV, rhesus rhadinovirus (RRV), was detected by PCR in sARL specimens. Although RRV was detected in nearly 80% of sARL specimens, frequency of virus infection among tumor cells was low. The incidence of RRV infection in PBMC rose with SIV coinfection, but RRV levels did not increase in SIV-infected animals that developed sARL, suggesting that RRV does not have a role in sARL development. This study also analyzes a spontaneous lymphoma in an SIV-negative chimpanzee (Pan troglodytes). Our results eliminate several possible cofactors in the development of this lymphoma, including familiar oncogenic mutations, and infection by chimpanzee-specific rhadinoviruses and lymphocryptoviruses. Finally, to study the influence of positive and negative growth factors in sARL, we used a sARL-derived cell line (LCL8664) that proliferated in response to human IL-6, but was sensitive to growth inhibition by human TGF-beta1. TGF-beta1 inhibited LCL8664 growth by downregulating expression of c-myc, inducing cell cycle arrest and increasing apoptosis. Treatment with IL-6 relieved TGF-beta1-mediated growth inhibition and apoptosis. IL-6 activated STAT3, ERK1/2 MAPK and P13K/Akt, and this activation was reduced by TGF-beta1 treatment. Among Bcl-2 family members, Mcl-1 expression was upregulated by IL-6, partially through the Pl-3K/Akt pathway. The responses of two human ARL-derived cell lines were examined for comparison: 2F7 (AIDS-related Burkitt's lymphoma) and UMCL01-101 (uncharacterized ARL). Like LCL8664, UMCL01-101 showed responsiveness to IL-6, inhibition by TGF-beta1, 2 and partial rescue of TGF-beta1-mediated inhibition by IL-6. Proliferation and protection from apoptosis mediated by IL-6 may provide an important survival signal for ARL cells in vivo , and may provide an important mechanism by which growth suppressive influences (e.g. TGF-beta1) in the tumor environment might be balanced / acase@tulane.edu

Hepatocellular carcinoma is one of the most common cancers in the world. Altered expression of a wide spectrum of genes has been associated with the development of hepatocellular carcinoma. However, the molecular mechanism(s) of hepatocarcinogenesis is not yet clear due to insufficient knowledge of the related genomic abnormalities. Using mRNA differential display technique, a 386 bp transcript was shown to be expressed at higher level in hepatocellular carcinoma as compared to the corresponding non-tumorous liver samples. Nucleotide sequence analysis revealed that this transcript had 99% similarity with the mRNA encoding human homogentisate 1,2-dioxygenase, the enzyme that converts homogentisate to maleylacetoacetate in the phenylalanine/tyrosine catabolic pathway. Northern analysis using HepG2 cell RNA further confirmed that this transcript and homogentisate 1,2-dioxygenase mRNA are identical. Elevated expression of homogentisate 1,2-dioxygenase mRNA was found in most of the tumors by RNA dot blot analysis. The activity of homogentisate 1,2-dioxygenase was also significantly elevated in hepatocellular carcinoma as compared to the non-tumorous liver while glyceraldehyde-3-phosphate dehydrogenase activity remained similar. To further study the involvement of homogentisate metabolism in hepatocarcinogenesis, the activity of fumarylacetoacetate hydrolase, the enzyme downstream of homogentisate 1,2-dioxygenase, converting fumarylacetoacetate into acetoacetate and fumarate, was investigated. Decreased activity of this enzyme was found in most of the tumors. Based on this small series of study, there was also a trend for increasing of the ratio of the activities of homogentisate 1,2-dioxygenase to fumarylacetoacetate hydrolase in tumor versus the non-tumorous liver in more advanced tumors. In the presence of homogentisate, the establishment of homogentisate metabolism in NIH 3T3 cells by the overexpression of homogentisate 1,2-dioxygenase led to several transformed phenotypes, i.e., decrease in population doubling time, increase in colony-formation efficiency and anchorage-independent growth in soft agar. These studies suggest that an abnormal homogentisate metabolism exists in the course of hepatocarcinogenesis and may play an important role during tumor formation. The results presented in this thesis will lead to a better knowledge of the genetic and epigenetic abnormalities related to hepatocellular carcinoma / acase@tulane.edu

The adenovirus E1A 243R oncoprotein induces DNA synthesis in quiescent cells. E1A-mediated transcriptional activation of cellular genes encoding essential DNA replication proteins is pivotal to the process of activating DNA synthesis. E1A modulates transcription directed by the promoter of the human proliferating cell nuclear antigen (PCNA) gene in a differential manner that is specified by distinct classes of cellular transcriptional activators. Specifically, E1A 243R stimulates or represses PCNA transcription activated by the glutamine-rich activator, Sp1Q, or the acidic activator, RegII, respectively. In the work described here, we explore mutants of E1A to characterize the mechanisms that lead to these diverse responses. Surprisingly, our findings indicate that an intact N-terminus of E1A is required for both activation and repression. These observations suggest that the N-terminus of E1A induces dramatically different effector-dependent transcriptional responses Substituting the PCNA basal promoter with an artificial basal promoter containing the adenovirus E1B gene TATA motif and upstream Gal4 binding sites (G5BCAT) alters the response to E1A 243R. We show here that the N-terminus of E1A mediates the differential transcriptional responses of G5BCAT. The basal promoter sequence from human immunodeficiency virus type 1 (HIV-1) represents a third class of basal promoter elements that responds to E1A modulation. To identify specific basal promoter elements that mediate differential responses to E1A, we prepared chimeras with motifs (TATA and initiator) from the HIV promoter introduced into the background of the PCNA basal promoter (-35 to +15). Although reduced transactivation was observed upon introduction of the HIV initiator motif into the PCNA basal promoter, a more dramatic decrease (from 8-fold to 2-fold) in transactivation was observed upon introduction of the HIV TATA motif. These observations suggest that basal promoter elements can function as selective determinants in response to transcriptional modulation by E1A 243R To identify DNA-protein complexes that mediate these transcriptional responses to E1A 243R, we employed electrophoretic mobility shift assays with the PCNA basal promoter and HeLa cell nuclear extracts. Two major complexes formed with PCNA basal promoter sequences (-35 to +15 relative to transcription initiation at +1), one that is located upstream (C2), and another that is situated around the initiation sequence (C1). Kinetic and competition experiments indicated that bacterially-expressed E1A 243R enhances the rate of complex formation and the stability of both complexes. These results suggest a molecular mechanism by which E1A transactivates the growth-responsive PCNA gene by stabilizing DNA-protein interactions on the TATA-less PCNA basal promoter. (Abstract shortened by UMI.) / acase@tulane.edu

Understanding the structure of mRNA is pivotal for performing studies on its biogenesis and functions. This statement can be illustrated by a study to evaluate the applicability of an Exon-Junction Hypothesis in conferring mRNA specificity in a reverse transcription-polymerase chain reaction (RT-PCR) assay. This hypothesis was tested on several mRNAs of human cytokines, including interleukin-1$\beta$, interleukin-2, interferon $\gamma$, interleukin-4, and interleukin-13. Approximately three-quarters of the exon-junction primers were found to be mRNA-specific. However, it became apparent that the yields of RT-PCR were affected by the secondary structure of mRNA A series of conceptual and experimental approaches were developed to help elucidate the overall architecture of mRNA. The total nucleotide sequence of mRNA was folded based on an algorithm of energy minimization. The folded RNA structure was interpreted into discrete segments of Closed Regions and Open Regions. A procedure known as Segment Analysis was developed to help visualize the Closed Regions and Open Regions on a folded RNA. The double stranded structure of Closed Region was composed of neighboring sequences which by themselves can be refolded into a structure identical to that on the overall folded RNA. The double stranded stems of open region was specified by long range interactions. The nucleotide sequence of an open region will not be refolded into a structure identical to those on the overall folded RNA For experimental verification of the predicted Closed Regions and Open Regions, a scanning approach and a regional approach were developed. For the scanning approach, a mRNA molecule was systematically scanned in an unbiased manner by a series of RT-PCR assays. Using hIL-1$\beta$ as a model system, an inverse proportional relationship was found between the specific yields of the RT-PCR assays and the free energy levels of the Close Regions covered by the amplified RNA segments For the regional approach, RT-PCR assays were designed to target directly either a Closed Region or an Open Region. Using hIL-10 mRNA as test system, a strong inverse proportional relationship was again observed between the specific yields of RT-PCR and the free energy levels of Closed Regions and Open Regions The knowledge gained on mRNA structure was then applied on developing specific and sensitive RT-PCR assays for a study of effects of interleukin-10 on Burkitt's lymphoma cells. The cytokines IL-4, IL-10, IL-13, and their receptors were found to express in both EBV-positive and EBV-negative Burkitt's lymphoma cells. In addition, recombinant human IL-10 enhanced growth of Raji cells; whereas neutralizing monoclonal antibody against human and viral IL-10 inhibited cell growth. These results supported an autocrine role for hIL-10. In addition, several candidate responsive genes of IL-10 have been identified in lymphoma cells by comparing the transcript profiles of the tumor cells The most important aspect of this thesis research was the development of approaches to study the structure of mRNA by a combination of structural prediction based on minimal energy and experimental verification by RT-PCR. These insights into mRNA secondary structure are not only useful for molecular biology research on RNA splicing, stability, RNA-protein interactions, and translation, but are also indispensable in exploring clinically oriented investigations such as gene diagnosis and genetic therapy. (Abstract shortened by UMI.) / acase@tulane.edu

Mixed-lineage leukemias are a group of hematologic malignancies that manifest as acute lymphoblastic, myeloid, or biphenotypic leukemias. They are caused by chromosomal translocations involving the MLL gene at the 11q23 locus, which lead to expression of chimeric proteins that fuse the N-terminus of the MLL protein to the C-terminus of one of more than fifty partners. MLL leukemia primarily develops in patients less than one year of age or in cases of therapy-related leukemia, and is associated with poor outcomes when treated with conventional cytotoxic drugs and bone marrow transplantation. Therefore, new molecular-based therapies are needed to target this group of leukemias The synthetic peptide PFWT was designed by our lab to disrupt the interaction between two common MLL fusion partners, AF4 and AF9. These proteins have been isolated as components of a transcription elongation complex whose other members include the RNA polymerase II regulator pTEFb and the chromatin modifying enzyme DOT1L. We have hypothesized that MLL fusions with AF4 or AF9 mistarget this complex to MLL target genes, whose expression contributes to the leukemia phenotype. We previously showed that PFWT demonstrates preferential toxicity toward MLL-rearranged leukemia cell lines. However, microarray studies did not identify any changes in gene expression of known MLL target genes in the sensitive cell line MV4-11 after PFWT treatment. Moreover, further experimentation has revealed that PFWT causes rapid cell membrane damage and actin cytoskeletal disruption in treated cells. We also demonstrate for the first time that the fusion partner AF9 is concentrated in the cytoplasm of the PFWT-sensitive cell line MV4-11. These findings have led us to propose a function for AF9 in actin cytoskeletal stability, which may also explain our observations with respect to the cytotoxic effects of the AF9-targeting peptide PFWT / acase@tulane.edu

No description available.