Gene regulation during placenta development: Murine adenosine deaminase as a model to study placenta specific expression

The formation of the placenta is one of the first and most important developmental events that occur in early mammalian embryogenesis. Even given this importance of the placenta, the academic community has largely ignored studying gene regulation during the development and maturation of the placenta. For this reason, an in-depth study of gene regulation in the trophoblast layer of the placenta using murine Adenosine Deaminase (Ada) as a model system has been undertaken. It has been determined that Ada is highly expressed in the placenta and is critical for embryo development. Dr. Kellems' laboratory has previously described a 1.8 kb fragment of the Ada 5 ′ flanking region that is capable of directing trophoblast specific expression in a transgenic model system. Preliminary studies have demonstrated several critical portions of this fragment that are necessary for the correct tissue specific expression in the placenta. My first specific aim was to elucidate the trans factor binding to one of these sequences, the FP3. Through electromobility shift assays (EMSA), the 30 bp FP3 was narrowed to a 5 bp sequence which computer databases predicted bound to Acute Myeloid Leukemia 1 (AML-1). This was confirmed by supershift analysis. The functional importance of this binding was demonstrated by a transgenic approach. A significant difference in expression of the reporter in the placenta was seen when the 5 bp sequence was mutated. This finding is a novel use for the AML-1 transcription factor which is the DNA binding portion of the heterodimer Core Binding Protein (CBP). The 5′ 240 bp region has also been demonstrated to contain functionally significant sequence. Through EMSA assays and computer predictions, the area has been narrowed to two pertinent regions that are predicted to contain GATA binding motifs. ^

The molecular mechanisms of sensing nucleoside analogue-induced DNA damage

Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematological malignancies. Upon being metabolized to their active triphosphate form, these agents are incorporated into DNA during replication or excision repair synthesis. Because DNA polymerases have a greatly decreased affinity for primers terminated by most nucleoside analogues, their incorporation causes stalling of replication forks. The molecular mechanisms that recognize blocked replication may contribute to drug resistance but have not yet been elucidated. Here, several molecules involved in sensing nucleoside analogue-induced stalled replication forks have been identified and examined for their contribution to drug resistance. ^ The phosphorylation of the DNA damage sensor, H2AX, was characterized in response to nucleoside analogues and found to be dependent on both time and drug concentration. This response was most evident in the S-phase fraction and was associated with an inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1-Cdc25A-Cdk2). Exposure of the Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), to cultures previously treated with nucleoside analogues caused increased apoptosis, clonogenic death, and a further log-order increase in H2AX phosphorylation, suggesting enhanced DNA damage. Ataxia-telangiectasia mutated (ATM) has been identified as a key DNA damage signaling kinase for initiating cell cycle arrest, DNA repair, and apoptosis while the Mre11-Rad50-Nbs1 (MRN) complex is known for its functions in double-strand break repair. Activated ATM and the MRN complex formed distinct nuclear foci that colocalized with phosphorylated H2AX after inhibition of DNA synthesis by the nucleoside analogues, gemcitabine, ara-C, and troxacitabine. Since double-strand breaks were undetectable, this response was likely due to stalling of replication forks. A similar DNA damage response was observed in human lymphocytes after exposure to ionizing radiation and in acute myelogenous leukemia blasts during therapy with the ara-C prodrug, CP-4055. Deficiencies in ATM, Mre11, and Rad50 led to a two- to five-fold increase in gemcitabine sensitivity, suggesting that these molecules contribute to drug resistance. Based on these results, a model is proposed for the sensing of nucleoside analogue-induced stalled replication forks that includes H2AX, ATM, and the Mre11-Rad50-Nbs1 complex. ^

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. ^

PRKCa: Identification of a Novel Downstream Target of WT1

Wilms tumor is a childhood tumor of the kidney arising from the undifferentiated metanephric mesenchyme. Tumorigenesis is attributed to a number of genetic and epigenetic alterations. In 20% of Wilms tumors, Wilms tumor gene 1 (WT1) undergoes inactivating homozygous mutations causing loss of function of the zinc finger transcription factor it encodes. It is hypothesized that mutations in WT1 result in dysregulation of downstream target genes, leading to aberrant kidney development and/or Wilms tumor. These downstream target genes are largely unknown, and identification is important for further understanding Wilms tumor development. Heatmap data of human Wilms tumor protein expression, generated by reverse phase protein assay analysis (RPPA), show significant correlation between WT1 mutation status and low PRKCα expression (p= 0.00013); additionally, p-PRKCα (S657) also shows decreased expression in these samples (p= 0.00373). These data suggest that the WT1 transcription factor regulates PRKCα expression, and that PRKCα plays a potential role in Wilms tumor tumorigenesis. We hypothesize that the WT1 transcription factor directly/indirectly regulates PRKCα and mutations occurring in WT1 lead to decreased expression of PRKCα. Prkcα and Wt1 have been shown to co-localize in E14.5 mesenchymal cells of the developing kidney. siRNA knockdown, in-vivo ablation, and tet-inducible expression of Wt1 each independently confirm regulation of Prkcα expression by Wt1 at both RNA and protein levels, and investigation into possible WT1 binding sites in PRKCα regulatory regions has identified multiple sites to be confirmed by luciferase reporter constructs. With the goal of identifying WT1 and PRKCα downstream targets, RPPA analysis of protein expression in mesenchymal cell culture, following lentiviral delivered shRNA knockdown of Wt1 and shRNA knockdown of Prkcα, will be carried out. Apart from Wilms tumor, WT1 also plays an important role in Acute Myeloid Leukemia (AML). WT1 mutation status has been implicated, controversially, as an independent poor-prognosis factor in leukemia, leading to decreased probability of overall survival, complete remission, and disease free survival. RPPA analysis of AML patient samples showed significant decreases in PRKCα/p-PRKCα protein expression in a subset of patients (Kornblau, personal communication); therefore, the possible role of WT1 and PRKCα in leukemia disease progression is an additional focus of this study. WT1 mutation analysis of diploid leukemia patient samples revealed two patients with mutations predicted to affect WT1 activity; of these two samples, only one corresponded to the low PRKCα expression cohort. Further characterization of the role of WT1 in AML, and further understanding of WT1 regulated PRKCα expression, will be gained following RPPA analysis of protein expression in HL60 leukemia cell lines with lentiviral delivered shRNA knockdown of WT1 and shRNA knockdown of PRKCα.

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^

Children with acute leukemia: A comparison of outcomes and cost-effectiveness from allogeneic blood stem cell and bone marrow transplantation

The relative merits of PBSCT versus BMT for children with standard and high risk hematologic malignancies remain unclear. In a retrospective single center study, we compared allogeneic peripheral blood stem cell transplantation (PBSCT) (n=30) with bone marrow transplantation (BMT) (n=110) in children with acute leukemia. We studied recipients of HLA matched sibling stem cells, and of stem cells from alternative donors (HLA mismatched and/or unrelated) and determined whether sourcing the stem cells from PB or marrow affected engraftment, incidence of acute and chronic GvHD, and disease-free survival at 1 year. Our results show a modest reduction in time to engraftment from PB stem cells and no greater risk of GvHD, but illustrate that the severity of the underlying disease is by far the greatest determinant of 1 year survival. Patients in the BMT group had a higher treatment success rate and lower costs than the recipients of the PBSCT within the standard but not the high risk disease group, where the treatment success rate and the cumulative costs were lower in the PBSCT group compared to the BMT group. Our current incremental cost-effectiveness ratio and analysis of uncertainty suggest that allogeneic transplantation of bone marrow grafts was a more cost-effective treatment option compared to peripheral blood stem cells in patients with standard risk childhood acute leukemia disease. For high risk disease our data are less prescriptive, since the differences were more limited and the range of costs much larger. Neither option demonstrated a clear advantage from a cost-effectiveness standpoint.^