p21 activated kinase 5 links multiple GTPase signaling pathways at mitochondria

The p21-activated kinase 5 (PAK5) is a serine/threonine protein kinase associated with the group 2 subfamily of PAKs. Although our understanding about PAK5 is very limited, it is receiving increasing interest due to its tissue specific expression pattern and important signaling properties. PAK5 is highly expressed in brain. Its overexpression induces neurite outgrowth in neuroblastoma cells and promotes survival in fibroblasts. ^ The serine/threonine protein kinase Raf-1 is an essential mediator of Ras-dependent signaling that controls the ERK/MAPK pathway. In contrast to PAK5, Raf-1 has been the subject of intensive investigation. However due to the complexity of its activation mechanism, the biological inputs controlling Raf-1 activation are not fully understood. ^ PAKs 1-3 are the known kinases responsible for phosphorylation of Raf-1 on serine 338, which is a crucial phosphorylation site for Raf-1 activation. However, dominant negative versions of these kinases do not block EGF-induced Raf-1 activation, indicating that other kinases may regulate the phosphorylation of Raf-1 on serine 338. ^ This thesis work was initiated to test whether the group 2 PAKs 4, 5 and 6 are responsible for EGF-induced Raf-1 activation. We found that PAK5, and to a lesser extent PAK4, can activate Raf-1 in cells. Our studies thereafter focused on PAK5. With the progress of our study we found that PAK5 does not significantly stimulate serine 338 phosphorylation of Triton X-100 soluble Raf-1. PAK5, however, constitutively and specifically associates with Raf-1 and targets it to a Triton X-100 insoluble, mitochondrial compartment, where PAK5 phosphorylates serine 338 of Raf-1. We further demonstrated that endogenous PAK5 and Raf-1 colocalize in Hela cells at the mitochondrial outer membrane. In addition, we found that the mitochondria-targeting of PAK5 is determined by its C-terminal kinase domain plus the upstream proximal region, and facilitated by the N-terminal p21 binding domain. We also demonstrated that Rho GTPases Cdc42 and RhoD associate with and regulate the subcellular localization of PAK5. Taken together, this work suggests that the mitochondria-targeting of PAK5 may link Ras and Rho GTPase-mediated signaling pathways, and sheds light on aspects of PAK5 signaling that may be important for regulating neuronal homeostasis. ^

Mechanism of IFN-induced apoptosis and sensitization by the proteasome inhibitor bortezomib in bladder cancer cells

Bladder cancer is the fifth most common cancer with more than 50,000 cases diagnosed each year. Interferon-α (IFNα) is mostly used in combination with BCG for the treatment of transitional cell carcinoma (TCC). To examine the effects of IFNα on bladder cancer cells, I analyzed a panel of 20 bladder cancer cell lines in terms of their sensitivity to IFNα-induced apoptosis and the underlying mechanisms. I identified three categories: cells that die after 48hr, after 72h, and cells resistant even after 72hr of IFNα treatment. Examination of the IFN-signal transduction pathway revealed that the defect was not due to abrogation of IFN signaling. Further analysis demonstrated dependency of IFN-induced apoptosis on caspase-8, implicating the role of death receptors in IFN-induced cell death. Of the six most-IFN-sensitive cell lines, the majority upregulated Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at the mRNA and protein level and IFN-induced cell death was mediated through TRAIL, while a minority of the most IFN-sensitive cells undergo apoptosis through a TNFα-dependent mechanism. IFNα resistance was due to either absence of TRAIL upregulation at the mRNA or protein level, resistance to exogenous rhTRAIL itself or lack of sensitization to IFN-induced cell death. Downregulation of XIAP, or XIAP inactivation through its regulator NFκB has been reported to sensitize tumor cells to death receptor-induced cell death. Baseline and IFN-inducible XIAP levels were examined in the most and least IFN-sensitive cells, knocking down XIAP and the p65 subunit of NFκB enhanced IFN-induced cell death, implicating XIAP downregulation as a mechanism through which bladder cancer cells are sensitized to IFN-induced apoptosis. To determine whether or not the proteasome inhibitor Bortezomib (BZ) sensitizes bladder cancer cells to IFN-induced cell death, the combined effects of IFN+BZ and the underlying molecular mechanisms were examined both in vitro and in vivo using two bladder xenograft models. In both models, tumor growth inhibition was the result of either increased cell death of tumor cells exerted by the two agents and/or inhibition of angiogenesis. In vitro, MAP downregulation in response to the combined treatment of IFN+BZ accounts for one of the mechanisms mediating IFN+BZ cell death in bladder cancer cells. ^

Developmental programming of tumor suppressor gene penetrance

Epidemiological studies have led to the hypothesis that major risk factors for developing diseases such as hypertension, cardiovascular disease and adult-onset diabetes are established during development. This developmental programming hypothesis proposes that exposure to an adverse stimulus or insult at critical, sensitive periods of development can induce permanent alterations in normal physiological processes that lead to increased disease risk later in life. For cancer, inheritance of a tumor suppressor gene defect confers a high relative risk for disease development. However, these defects are rarely 100% penetrant. Traditionally, gene-environment interactions are thought to contribute to the penetrance of tumor suppressor gene defects by facilitating or inhibiting the acquisition of additional somatic mutations required for tumorigenesis. The studies presented herein identify developmental programming as a distinctive type of gene-environment interaction that can enhance the penetrance of a tumor suppressor gene defect in adult life. Using rats predisposed to uterine leiomyoma due to a germ-line defect in one allele of the tuberous sclerosis complex 2 (Tsc-2) tumor suppressor gene, these studies show that early-life exposure to the xenoestrogen, diethylstilbestrol (DES), during development of the uterus increased tumor incidence, multiplicity and size in genetically predisposed animals, but failed to induce tumors in wild-type rats. Uterine leiomyomas are ovarian-hormone dependent tumors that develop from the uterine myometrium. DES exposure was shown to developmentally program the myometrium, causing increased expression of estrogen-responsive genes prior to the onset of tumors. Loss of function of the normal Tsc-2 allele remained the rate-limiting event for tumorigenesis; however, tumors that developed in exposed animals displayed an enhanced proliferative response to ovarian steroid hormones relative to tumors that developed in unexposed animals. Furthermore, the studies presented herein identify developmental periods during which target tissues are maximally susceptible to developmental programming. These data suggest that exposure to environmental factors during critical periods of development can permanently alter normal physiological tissue responses and thus lead to increased disease risk in genetically susceptible individuals. ^

The roles of UPF3a and UPF3b in nonsense mediated decay

Nonsense-mediated decay (NMD) degrades aberrant transcripts containing premature termination codons (PTCs). The T-cell receptor (TCR) locus undergoes error-prone rearrangements that frequently acquire PTCs. Transcripts harboring PTCs from this locus are downregulated much more than transcripts from non-rearranging genes. Efficient splicing is essential for this robust downregulation. ^ Here I show that TCR NMD is unique in another respect: it is not impaired by RNAi-mediated depletion of the NMD factor UPF3b. This differentiates TCR transcripts from classical NMD (assayed using β-globin or triose phosphate isomerase transcripts), which does depend on UPF3b. Depletion of UPF3a, which encodes a gene related to UPF3b, also had no effect on TCR NMD. Mapping experiments identified TCR sequences that when deleted or mutated caused a switch to UPF3b dependence. Since UPF3b dependence was invariably accompanied by less efficient RNA splicing, this suggests that UPF3b-dependent NMD occurs when transcripts are generated by inefficient splicing. Microarray analysis revealed the existence of many NMD-targeted mRNAs from wild-type genes whose downregulation is impervious to UPF3b depletion. This suggests the existence of an alternative NMD pathway independent of UPF3b that is widely used to downregulate the level of both normal and mutant transcripts. ^ During the course of my studies, I also found that the function of UPF3a is fundamentally distinct from that of UPF3b in several aspects. First, classical NMD failed to be impaired by UPF3a depletion, whereas it was reversed by UPF3b depletion. Second, UPF3a depletion had no effect on NMD elicited by tethered UPF2, whereas UPF3b depletion blocked this response. Thus, UPF3a does not function in classical NMD. Third, UPF3b depletion upregulated the expression of UPF3a, whereas UPF3a depletion had no effect on UPF3b expression. This suggests that a UPF3b-mediated feedback network exists that regulates the UPF3a expression. Lastly, UPF3a depletion but not UPF3b depletion significantly upregulated TCR precursor RNAs. This suggests that UPF3a, not UPF3b, functions in the surveillance of precursor RNAs, which typically contain many PTCs in the introns. Collectively, my data suggests that UPF3a and UPF3b are not functionally redundant, as previously thought, but instead have separable functions. ^

The role of deregulated E2F1 expression in brain tumors

Mutations disabling the retinoblastoma (Rb) pathway are among the most common in human cancers, including brain cancer. These mutations promote tumor development through deregulated control of the E2F family of transcription factors. E2F1 belongs to a class of E2F's identified as transcriptional activators and involved in the G1/S phase transition of the cell. However, E2F-1 presents with a paradox as it is considered to have membership in two gene classes, functioning as both an oncogene and a tumor suppressor. This unusual trait generates a degree of uncertainty on the role that E2F1 plays in the development or maintenance of any given tumor. Here we show that E2F1 functions as an oncogene in brain tumors through the generation of mice engineered to overexpress E2F1 specifically within glial cells and neuronal progenitors as directed by the GFAP promoter. Mice carrying the transgene develop with high penetrance a phenotype characterized by neurological deficits including paresia, ataxia, head tilt and seizures. MRI imagining of the tgE2F1 mice reveals a low incidence of mild hydrocephalus, and most notably, histological analysis demonstrates that 25% of tgE2F1 mice present with the spontaneous formation of malignant brain tumors. Overall these neoplasms show histological features from a wide range of aggressive brain cancers including medulloblastoma, choroid plexus carcinoma, primary neuroectodermic tumor and malignant gliomas. Isolation and characterization of astrocytes from the tgE2F1 animal reveals a highly proliferative population of cells with 55% ± 2.5 of the tgE2F1astrocytes, 35% ± 3.4 normal mouse astrocytes in S-phase and the acquired capacity to grow in anchorage independent conditions. Additionally tgE2F1 astrocytes show an aberrant phenotype with random chromosomal fusions and nearly all cells demonstrating polyploidy. Taken together, this model forces a comparison to human brain tumor formation. Mouse age as related to tumoral mimics the human scenario with juvenile tgE2F1 mice presenting embryonal tumors typically identified in children, and older tgE2F1 mice demonstrating gliomas. In this regard, this study suggests a global role for E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain. ^

Investigation of the molecular mechanisms of endometrial hyperplasia

While there is considerable information on the molecular aberrations associated with the development of endometrial cancer, very little is known of changes in gene expression associated with its antecedent premalignant condition, endometrial hyperplasia. In order to address this, we have compared the level of expression of components of the IGF-I signaling pathway in human endometrial hyperplasia to their level of expression in both the normal pre-menopausal endometrium and endometrial carcinoma. We have also characterized the molecular characteristics of endometrial hyperplasia as it occurs in a murine model of hormone-dependent tumorigenesis of the female reproductive tract. ^ There was a significant and selective increase in the expression of the IGF-I Receptor (IGF-IR) in both human hyperplasia and carcinoma as compared to the normal endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in hyperplasia and carcinoma there is activation of the downstream component Akt. The expression of the PTEN tumor suppressor is decreased in a subset of subjects with hyperplasia and in all of the carcinomas. The simultaneous loss of PTEN expression and increased IGF-IR activation in the hyperplastic endometrium was associated with an increased incidence of endometrial carcinoma elsewhere within the uterus. In the rodent hyperplasia, there was a significant increase in the expression and activation of Akt that appears to be attributable to a marked increase in the expression of IGF-II. ^ Our studies have demonstrated the pathologic proliferation of both the human and rodent endometrium is linked to a marked activation of the Akt pathway. However the cause of this dysregulation is different in the human disease and the animal model. In rodents, hyperplasia is linked to increased expression of one of the ligands of the IGF-IR, IGF-II. In humans the IGF-I receptor itself is upregulated and activated. Additional activation of the Akt pathway via the suppression of PTEN activity, results in conditions that are associated with the marked increase in the probability of developing endometrial cancer. Our data suggests that increased activity of the IGF-I pathway plays the key role in the hyperproliferative state characteristic of endometrial hyperplasia and cancer.^

Lethal giant discs, a novel C2 domain protein, restricts Notch activation during endocytosis

The Notch signaling pathway plays a central role in metazoan growth and patterning, and its deregulation leads to many human diseases, including cancer. It is therefore important to understand the modes of Notch signaling regulation. Recent discoveries have demonstrated that mutations in conserved endosomal pathway components such as Erupted and Vps25 can ectopically activate Notch signaling in Drosophila. Mutations in the tumor suppressor lethal giant discs (lgd) display similar but even stronger and more specific Notch activation than in the erupted and vps25 mutant animals. This Notch activation in lgd mutant tissues causes hyperplastic overgrowth of the Drosophila imaginal discs, and the eventual lethality of the animal. However, the gene that encodes Lgd, and its function in the Notch pathway have not yet been identified. ^ I have found that Lgd is a novel, conserved C2 domain protein that regulates Notch trafficking. Lgd cell-autonomously restricts Notch signaling in the Drosophila wing disc to the target cells in the D/V boundary. The function of Lgd lies at or upstream of Notch S3 activation, but Lgd doesn't affect the binding affinities between Notch and Delta. Lgd is also not required for cis-inhibition of Notch signaling by ligands. Notch accumulates on the early endosome in lgd mutant cells and signals in a ligand-independent manner, a result that has previously been seen in endosomal pathway mutants. Interestingly, Notch activation in lgd mutant cells is dependent on the endosomal protein Hrs, and Lgd activity appears to be downstream of Hrs function in endocytosis. Taken together, my data identify Lgd as a novel tumor suppressor protein that regulates Notch signaling by targeting Notch for degradation or recycling. ^

Nuclear functions of dynein light chain 1 in hormone action

It is widely accepted that the process of breast cancer tumorigenesis involves estrogen receptor-alpha (ER)-regulated stimulatory pathways, which feed into survival, cell cycle progression and proliferative response. Recent data from Kumar laboratory indicate that dynein light chain 1 (DLC1) plays a role in survival, motility and invasiveness, all of which are required for a successful tumorigenesis process. In the present research, we have discovered a mechanistic bidirectional regulatory link between the DLC1 and ER. We found that DLC1 facilitates ligand-induced ER transactivation involving the recruitment of the DLC1-ER complex to ER-target genes. To gain insights into the mechanism by which DLC1 regulates the ER pathway, we set out to identify novel DLC1-interacting proteins. Among other proteins, we identified KIBRA and Ciz1 as two novel DLC1-interacting proteins. We found that the KIBRA-DLC1 complex is recruited to ER-responsive promoters, and that KIBRA-DLC1 interaction is needed for the recruitment of ER to its targets as well as for ER's transactivation function. Finally, we found that KIBRA utilizes its histone H3interacting glutamic acid-rich region to regulate the transactivation activity of ER. During the course of this work, we also discovered that DLC1 interacts with Cdk2 and Ciz1, and such interactions play a direct accelerating role in the G1-S transition of breast cancer cells. While delineating the role of Ciz1 in hormone-responsive cancer cells, we found that Ciz1 is an estrogen-responsive gene, and acts as a co-regulator of ER. Accordingly, Ciz1 overexpression in breast cancer cells conferred estrogen hypersensitivity, promoted the growth-rate, anchorage-independency and tumorigenic properties. Collectively, findings made during the course of the present dissertation research introduced two new molecular players in the action of ER in breast cancer cells, with a particular focus on cell cycle progression and ER-chromatin target regulation. In addition, findings presented here provide novel mechanistic insight about the contribution of DLC1 and its interacting proteins in amplifying the hormone action and promoting the process of breast cancer tumorigenesis. ^

Transcriptional upregulation of the p21Cip1 promoter by STAT3 and nuclear ErbB2

Over-expression of the receptor tyrosine kinase ErbB2 is prevalent in approximately 30% of human breast carcinomas and confers Taxol resistance. In breast cancer cells, Taxol induces tubulin polymerization and hyperstable microtubule formation. This in turn prematurely activates Cdc2 kinase allowing early entry into the G2/M phase of the cell cycle resultant in mitotic catastrophe followed by apoptosis. Over-expression of ErbB2 upregulates p21Cip1, which inhibits Cdc2 activation, and leads to Taxol resistance in patients. However, the mechanism of ErbB2-mediated p21 Cip1 upregulation is unclear. Here in this study, we investigated the mechanism of ErbB2 downstream signaling events leading to upregulation. The CDKN1A (p21Cip1) gene promoter contains numerous cis-elements including a Signal transducer and activator of transcription (STAT) Inducable Element (SIE) located at -679 kb. Our studies showed ErbB2 overexpressing cells had increased activated levels of STAT3, and therefore we hypothesized that STAT3 is responsible for the upregulation of the p21Cip1 promoter by ErbB2. EMSA and ChIP assays confirmed the binding of STAT3 to the p21Cip1 promoter and luciferase assays showed higher p21 Cip1 promoter activity in ErbB2 over-expressing transfectants when compared to parental cells, in a STAT3 binding site dependant manner. Additionally, reduced level of STAT3 led to reduced p21Cip1 protein expression and promoter activity indicating that both the STAT3 binding site and STAT3 protein are required for ErbB2-mediated p21Cip1 upregulation. Further investigation of ErbB2 downstream signaling showed increased Src kinase activity in ErbB2 over-expressing cells which was required for ErbB2-mediated STAT3 activation and p21Cip1 increase. Treatment of ErbB2 over-expressing resistant cells with STAT3 inhibitor peptides sensitized the cells to Taxol. In addition to classical signal transduction pathways, I identified a novel ErbB2 mediated regulatory mechanism of p21Cip1. I found that a nuclear ErbB2 and STAT3 complex binds directly to the p21Cip1 promoter offering a non-classical mechanism of p21Cip1 promoter regulation. These data suggest that ErbB2 over-expression can confer Taxol resistance of breast cancer cells by transcriptional upregulation of p21 Cip1 via activation of STAT3 by Src kinase and also by cooperation with nuclear ErbB2. The data suggest a potential clinical mechanism for STAT3 inhibitors in sensitizing ErbB2 over-expressing breast cancers to Taxol. ^

Caenorhabditis elegans germinal center kinase (Gck-1) is a p-body component that inhibits precocious ectopic MAP kinase dependent meiotic maturation

The Caenorhabditis elegans germline is an excellent model system for studying meiosis, as the gonad contains germ cells in all stages of meiosis I prophase in a linear temporal and spatial pattern. To form healthy gametes, many events must be coordinated. Failure of any step in the process can reduce fertility. Here, we describe a C. elegans Germinal Center Kinase, GCK-1, that is essential for the accurate progression of germ cells through meiosis I prophase. In the absence of GCK-1, germ cells undergo precocious maturation due to the activation of a specific MAP kinase isoform. Furthermore, GCK-1 localizes to P-bodies, RNP particles that have been implicated in RNA degradation and translational control. Like two other components of C. elegans germline P-bodies, GCK-1 functions to limit physiological germ cell apoptosis. This is the first study to identify a role for a GCK-III kinase in metazoan germ cell development and to link P-body function with MAP kinase activation and germ cell maturation. ^

The role of coactivator associated arginine methyltransferase 1 (CARM1) in nuclear receptor mediated transcription

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARMI/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we generated and characterized CARM1 knockout mice. Embryos with a targeted disruption of CARM1 are 35% smaller in size than the wild-type littermates and die perinatally. We also generated Carm1-/- and Carm1+/+ mouse embryonic fibroblasts and tested gene expression in response to estrogen. Estrogenresponsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. We subsequently studied the role of CARM1 in estrogen signaling in viva in the mammary gland. Conditional knockout of CARM1 in mammary gland and Carml-1-embryonic mammary anlagen transplant experiments did not show any defects in growth and development of the glands. To further dissect the role of CARM1 in estrogen receptor mediated transactivation, we performed cDNA microarray and serial analysis of gene expression on Carm1-/- and Carm1+/+ embryos treated with the estrogen analog, DES. Our results indicate global changes in estrogen regulated genes as well as genes involved in lipid homeostasis. Marker genes for Peroxisome Proliferator Activated Receptor γ (PPARγ) activity, adipsin and aP2, are downregulated in the Carm1-/- embryos. Furthermore, OCT frozen sections of 18.5dpc embryos, processed simultaneously for oil red O staining to look for neutral fat, reveals greatly reduced brown fat accumulation in the Carm1-/- embryos in contrast to wild-type and gain-of-function Carm1 transgenic (ubiquitous) embryo. We used a well-established 3T3-L1 preadipocyte cell line to knockdown CARM1 by short hairpin RNA. 3T3-L1 cells with CARM1 knockdown showed greatly reduced potential to differentiate into mature lipid accumulating adipocytes upon administration of adipogenic stimuli. Ligand-dependent activation of reporter genes by the PPARγ receptor showed that PPRE-luciferase reporter activity was enhanced in the presence of CARM1, additionally, luciferase activity was reduced to background levels when enzyme dead CARM1 (CARM1-VLD) was used. Thus, in this study, we have identified novel pathways that use CARM1 as coactivator and showed that CARM1 functions as a key component of PPARγ receptor mediated gene expression. ^

Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^

Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

Regulation of RNA splicing by nonsense mutations

Translation termination as a result of premature nonsense codon-incorporation in a RNA transcript can lead to the production of aberrant proteins with gain-of-function or dominant negative properties that could have deletrious effects on the cell. T-cell Receptor (TCR) genes acquire premature termination codons two-thirds of the time as a result of the error-prone programmed rearrangement events that normally occur during T-cell development. My studies have focused on the fate of TCR precursor mRNAs in response to in-frame nonsense mutations. ^ Previous published studies from our laboratory have shown that TCR precursor mRNAs are subject to nonsense mediated upregulation of pre-mRNA (NMUP). In this dissertation, I performed substitution and deletion analysis to characterize specific regions of TCR which are required to elicit NMUP. I performed frame- and factor-dependence studies to determine its relationship with other nonsense codon induced responses using several approaches including (i) translation dependence studies (ii) deletion and mutational analysis, as well as (iii) siRNA mediated knockdown of proteins involved. I also addressed the underlying molecular mechanism for this pre-mRNA upregulation by (i) RNA half-life studies using a c-fos inducible promoter, and (ii) a variety of assays to determine pre-mRNA splicing efficiency. ^ Using these approaches, I have identified a region of TCR that is both necessary and sufficient to elicit (NMUP). I have also found that neither cytoplasmic translation machinery nor the protein UPF1 are involved in eliciting this nuclear event. I have shown that the NMUP can be induced not only by nonsense and frameshift mutations, but also missense mutations that disrupt a cis splicing element in the exon that contains the mutation. However, the effect of nonsense mutations on pre-mRNA is unique and distinguishable from that of missense mutations in that nonsense mutations can upregulate pre-mRNA in a frame-dependent manner. Lastly, I provide evidence that NMUP occurs by a mechanism in which nonsense mutations inhibit the splicing of introns. In summary, I have found that TCR precursor mRNAs are subject to multiple forces involving both RNA splicing and translation that can either increase or decrease the levels of these precursor mRNAs. ^

Identification and characterization of distinct classes of cAMP-pulse-induced genes in Dictyostelium discoideum development

The unicellular amoeba Dictyostelium discoideum embarks on a developmental program upon starvation. During development, extracellular oscillatory cAMP signaling orchestrates the chemotaxis-mediated aggregation of ∼105 amoebae and is required for optimal induction of so-called pulse-induced genes. This requirement for pulsatile CAMP reflects adaptation of the cAMP-receptor-mediated pathways that regulate these genes. Through examination of a collection of pulse-induced genes, we defined two distinct gene classes based on their induction kinetics and the impact of mutations that impair PKA signaling. The first class (represented by D2 and prtA) is highly dependent on PKA signaling, whereas the second class (represented by carA, gpaB, and acaA) is not. Analysis of expression kinetics revealed that these classes are sequentially expressed with the PKA-independent genes peaking in expression before the PKA-dependent class. Experiments with cycloheximide, an inhibitor of translation, demonstrated that the pulse induction of both classes depends on new protein synthesis early in development. carA and gpaB also exhibit pulse-independent, starvation-induced expression which, unlike their pulse induction, was found to be insensitive to cycloheximide added at the outset of starvation. This result indicates that the mechanism of starvation induction pre-exists in growing cells and is distinct from the pulse induction mechanism for these genes. In order to identify cis-acting elements that are critical for induction of carA, we constructed a GFP reporter controlled by a 914-base-pair portion of its promoter and verified that its expression was PKA-independent, pulse-inducible, and developmentally regulated like the endogenous carA gene. By a combination of truncation, internal deletion, and site-directed mutation, we defined several distinct functional elements within the carA promoter, including a 39-bp region required for pulse induction between base pairs -321 and -282 (relative to the transcription start site), a 131-bp region proximal to the start site that is sufficient for starvation induction, and two separate enhancer domains. Identification of factors that interact with these promoter elements and genetic approaches exploiting the GFP reporter described here should help complete our understanding of the mechanisms regulating these genes, including adaptation mechanisms that likely also govern chemotaxis of Dictyostelium and mammalian cells. ^