The biology and mechanism of chemoresistance of brain metastases

Primary brain neoplasms and metastases to the brain are generally resistant to systemic chemotherapy. The purpose of theses studies was to determine the mechanism(s) for this resistance. We have developed a model to study the biology of brain metastasis by injecting metastatic K1735 melanoma cells into the carotid artery of syngeneic C3H/HeN or nude mice. The resulting brain lesions are produced in the parenchyma of the brain. Mice with subcutaneous or brain melanoma lesions were treated intravenously with doxorubicin (DXR) (7 mg/kg). The s.c. lesions regressed in most of the mice whereas no therapeutic benefits were produced in mice with brain metastases. The intravenous injection of sodium fluorescine revealed that the blood-brain barrier (BBB) is intact in and around brain metastases smaller than 0.2 mm$\sp2$ but not in larger lesions, implying that the BBB is not a major obstacle for chemotherapy of brain metastases.^ Western blot and FACS analyses revealed that K1735 melanoma brain metastases expressed high levels of P-glycoprotein (P-gp) as compared to s.c. tumors or in vitro cultures. Similarly, K1735 cells from brain metastases expressed higher levels of mdrl mRNA. This increased expression of mdrl was due to adaptation to the local brain environment. We base this conclusion on the results of two studies. First, K1735 cells from brain metastases cultured for 7 days lost the high mdrl expression. Second, in crossover experiments K1735 cells from s.c. tumors (low mdrl expression) implanted into the brain exhibited high levels of mdrl expression whereas cells from brain metastases implanted s.c. lost the high level mdrl expression.^ To investigate the mechanism by which the brain environment upregulates mdrl expression of the K1735 cells we first studied the regulation of P-gp in brain endothelial cells. Since astrocytes are closely linked with the BBB we cocultured brain endothelial cells for 3 days with astrocytes. These endothelial cells expressed high levels of mdrl mRNA and protein whereas endothelial cells cocultured with endothelial cells or fibroblasts did not. We next cocultured K1735 melanoma cells with astrocytes. Here again, astrocytes (but not fibroblasts or tumor cells) uprelated the mdrl expression in K1735 tumor cells. This upregulation inversely correlated with intracellular drug accumulation and sensitivity to DXR.^ The data conclude that the resistance of melanoma brain metastases to chemotherapy is not due to an intact BBB but to the upregulation of the mdrl gene by the organ microenvironment, i.e., the astrocytes. This epigenetic mediated resistance to chemotherapy has wide implications for the therapy of brain metastases. ^

Molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$-adrenergic receptor

The $\beta$-adrenergic receptor ($\beta$AR), which couples to G$\sb{\rm s}$ and activates adenylylcyclase, has been a prototype for studying the activation and desensitization of G-protein-coupled receptors. The main objective of the present study is to elucidate the molecular mechanisms of protein kinase-mediated desensitization and internalization of the $\beta$AR.^ Activation of cAPK or PKC causes a rapid desensitization of $\beta$AR stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the $\beta$AR, RRSSK$\sp{263}$. To determine the role of the individual serines in the cAPK- and PKC-meditated desensitizations, wild type (WT) and mutant $\beta$ARs containing the substitutions, Ser$\sp{261} \to$ A, Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and Ser$\sp{261/262} \to$ A, were constructed and stably transfected into L cells. The cAPK-mediated desensitization was decreased 70-80% by the Ser$\sp{262} \to$ A, Ser$\sp{262} \to$ D, and the Ser$\sp{261/262} \to$ A mutations, but was not altered by the Ser$\sp{261} \to$ A substitution, demonstrating that Ser$\sp{262}$ was the primary site of the cAPK-induced desensitization. The PMA/PKC-induced desensitization was unaffected by either of the single serine to alanine substitutions, but was reduced 80% by the double serine to alanine substitution, suggesting that either serine was sufficient to confer the PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser$\sp{262} \to$ A, was partially uncoupled. The Ser$\sp{262} \to$ D mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor.^ To accomplish the in vivo phosphorylation of the $\beta$AR, we used two epitope-modified $\beta$ARs, hemagglutinin-tagged $\beta$AR (HA-$\beta$AR) and 6 histidine-tagged $\beta$AR (6His-$\beta$AR), for a high efficiency purification of the $\beta$AR. Neither HA-$\beta$AR nor 6His-$\beta$AR altered activation and desensitization of the $\beta$AR significantly as compared to unmodified wild type $\beta$AR. 61% recovery of ICYP-labeled $\beta$AR was obtained with Ni-NTA column chromatography.^ The truncation 354 mutant $\beta$AR(T354), lacking putative $\beta$ARK site(s), displayed a normal epinephrine stimulation of adenylylcyclase. Although 1.0 $\mu$M epinephrine induced 60% less desensitization in T354 as compared to wild type $\beta$AR, 1.0 $\mu$M epinephrine-mediated desensitization in T354 was 35% greater than PGE$\sb1$-mediated desensitization, which is essentially identical in both WT and T354. These results suggested that sequences downstream of residue 354 may play a role in homologous desensitization and that internalization may be attributed to the additional desensitization besides the cAMP mechanism in T354 $\beta$AR. (Abstract shortened by UMI.) ^

Molecular mechanisms of prostatic carcinoma growth and progression

Prostate cancer represents the most commonly diagnosed malignancies in American men and is the second leading cause of male cancer deaths. The overall objectives of this research were designed to understand the cellular and molecular mechanisms of prostatic carcinoma growth and progression. This dissertation was divided into two major parts: (1) to clone and characterize soluble factor(s) associated with bone that may mediate prostatic carcinoma growth and progression; (2) to investigate the roles of extracellular matrix in prostatic carcinogenesis.^ The propensity of prostate cancer cells to metastasize to the axial skeleton and the subsequent osteoblastic reactions observed in the bone indicate the possible reciprocal cellular interaction between prostate cancer cells and the bone microenvironment. To understand the molecular and cellular basis of this interaction, I focused on the identification and cloning of soluble factor(s) from bone stromal cells that may exert direct mitogenic action on cultured prostate cells. A novel BPGF-1 gene expressed specifically by bone and male accessory sex organs (prostate, seminal vesicles, and coagulating gland) was identified and cloned.^ The BPGF-1 was identified and cloned from a cDNA expression library prepared from a human bone stromal cell line, MS. The conditioned medium (CM) of this cell line contains mitogenic materials that induce human prostate cancer cell growth both in vivo and in vitro. The cDNA expression library was screened by an antibody prepared against the mitogenic fraction of the CM.^ The cloned BPGF-1 cDNA comprises 3171 nucleotides with a single open reading frame of 1620 nucleotides encoding 540 amino acids. The BPGF-1 gene encodes two transcripts (3.3 and 2.5 kb) with approximately equal intensity in human cells and tissues, but only one transcript (2.5 kb) in rat and mouse tissues. Southern blot analysis of human genomic DNA revealed a single BPGF-1 gene. The BPGF-1 gene is expressed predominantly in bone and seminal vesicles, but at a substantially lower level in prostate. Polyclonal antibodies generated from synthetic peptides that correspond to the nucleotide sequences of the cloned BPGF-1 cDNA reacted with a putative BPGF-1 protein with an apparent molecular weight of 70 kDa. The conditioned media isolated from COS cells transfected with BPGF-1 cDNA stimulated the proliferation and increased the anchorage-independent growth of prostate epithelial cells. These findings led us to hypothesize that BPGF-1 expression in relevant organs, such as prostate, seminal vesicles, and bone, may lead to local prostate cancer growth, metastasis to the seminal vesicles, and subsequently dissemination to the skeleton.^ To assess the importance of extracellular matrix in prostatic carcinogenesis, the role of extracellular matrix in induction of rat prostatic carcinoma growth in vivo was evaluated. NbE-1, a nontumorigenic rat prostatic epithelial cell line, was induced to form carcinoma in athymic nude hosts by coinjecting them with Matrigel and selected extracellular matrix components. Induction of prostatic tumor formation by laminin and collagen IV was inhibited by their respective antibodies. Prostatic epithelial cells cloned from the tumor tissues were found to form tumors in athymic nude hosts in the absence of exogenously added extracellular matrix. These results suggest that extracellular matrix induce irreversibly prostatic epithelial cells that behave distinctively different from the parental prostatic epithelial cell line. ^

Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

Population and molecular evolution studies on the Melanocortin 1 receptor locus implicate its role in human pigmentation variation

Human pigmentation is a complex trait with the observed variation caused by the varied production of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) by the melanocytes. The melanocortin 1 receptor (MC1R), a G protein-coupled receptor expressed in the melanocytes, is a regulator eu- and phaeomelanin synthesis, and MC1R mutations causing skin and coat color changes are known in many mammals. To understand the role of MC1R in human pigmentation variation, I have sequenced the MC1R gene in 121 individuals sampled from world populations. In addition, I have sequenced the MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon to study the evolution of MC1R and to infer the ancestral human MC1R sequence. The ancestral MC1R sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. To further evaluate the role of MC1R variants in human pigmentation variation, I have combined these molecular evolution and population studies with functional assays on MC1R variants and primate MC1Rs. ^

A study in the molecular and cellular functions of GSK3beta in prostate adenocarcinoma

Kinases are part of a complex network of signaling pathways that enable a cell to respond to changes in environmental conditions in a regulated and coordinated way. For example, Glycogen Synthase Kinase 3 beta (GSK3β) modulates conformational changes, protein-protein interaction, protein degradation, and activation of unique domains in proteins that transduce signals from the extracellular milieu to the nucleus. ^ In this project, I investigated the expression and function that GSK3β exhibits in prostate cells. The capacity of GSK3β to regulate two transcription factors (JUN and CREB), which are known to be inversely utilized in prostate tumor cells, was measured. JUN/AP1 is constitutively activated in PC-3 cells; whereas, CREB/CRE activity is ∼20 fold less than the former. GSK3β overexpression obliterates JUN/AP1 activity. With respect to CREB GSK3β increases CREB/CRE activity. Cellular levels of active GSK3β can determine whether JUN or CREB is preferentially active in the PC-3s. Theoretically, in response to a particular cellular context or stimulus, a cell may coordinate JUN and CREB function by regulating GSK3β.^ A comparison of various prostate cell lines showed that active GSK3β is less expressed in normal prostate epithelial cells than in tumor cells. Differentially expressed active (GSK3β) may correlate with progression of prostate carcinoma. If a known marker associated with carcinoma of the prostate could be shown to be regulated by GSK3β then, further study of GSK3β may lead to a better understanding of both possible prevention of the disease and improved therapy for advanced stages. ^ The androgen receptor (AR) is an intriguing phosphoprotein whose regulation is potentially determined by a variety of kinases. One of these is (GSK3β) I found that (GSK3β) is a regulator of the androgen receptor in both the unliganded and liganded states. It can inhibit AR function as measured by reporter assays. Also, GSK3β associates with the AR at the DNA binding domain because deletion constructs expressing either the n-terminus or the c-terminus (both having the DBD in common) immunoprecipitated with GSK3β. Increased understanding of how GSK3β functions in prostate cancer would provide clues into how (1) certain signal pathways are coordinated and (2) the androgen receptor may be regulated. ^

Molecular mechanisms of NF-kappaB activation in metastatic pancreatic cancer cells

Pancreatic adenocarcinoma is the fourth leading cause of adult cancer death in the United States. At the time of diagnosis, most patients with pancreatic cancer have advanced and metastatic disease, which makes most of the traditional therapeutic strategies are ineffective for pancreatic cancer. A better understanding of the molecular basis of pancreatic cancer will provide the approach to identify the new strategies for early diagnosis and treatment. NF-κB is a family of transcription factor that play important roles in immune response, cell growth, apoptosis, and tumor development. We have shown that NF-κB is constitutively activated in most human pancreatic tumor tissues and cell lines, but not in the normal tissues and HPV E6E7 gene-immortalized human pancreatic ductal epithelial cells (HPDE/E6E7). By infecting the pancreatic cancer cell line Aspc-1 with a replication defective retrovirus expressing phosphorylation-defective IκBα (IκBαM), the constitutive NF-κB activation is blocked. Subsequent injection of this Aspc-1/IκBαM cells into the pancreas of athymic nude mice showed that liver metastasis is suppressed by the blockade of NF-κB activation. Current studies showed that an autocrine mechanism accounts for the constitutive activation of NF-κB in metastatic human pancreatic cancer cell lines, but not in nonmetastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1α (IL-1α) was the primary cytokine secreted by these cells that activates NF-κB. Inhibition of IL-1α activity suppressed the constitutive activation of NF-κB and the expression of its downstream target gene, uPA, in metastatic pancreatic cancer cell lines. Even though IL-1α is one of the previously identified NF-κB downstream target genes, our results demonstrate that regulation of IL-1α expression is independent of NF-κB and primarily dependent on AP-1 activity, which is in part induced by overexpression of EGF receptors and activation of MAP kinases. In conclusion, our findings suggest a possible mechanism by which NF-κB is constitutively activated in metastatic human pancreatic cancer cells and a possible missing mechanistic links between inflammation and cancer. ^

Insights into the biology of lymphatic metastasis

Recent data suggest that the generation of new lymphatic vessels (i.e. lymphangiogenesis) may be a rate-limiting step in the dissemination of tumor cells to regional lymph nodes. However, efforts to study the cellular and molecular interactions that take place between tumor cells and lymphatic endothelial cells have been limited due to a lack of lymphatic endothelial cell lines available for study. ^ I have used a microsurgical approach to establish conditionally immortalized lymphatic endothelial cell lines from the afferent mesenteric lymphatic vessels of mice. Characterization of lymphatic endothelial cells, and tumor-associated lymphatic vessels revealed high expression levels of VCAM-1, which is known to facilitate adhesion of some tumor cells to vascular endothelial cells. Further investigation revealed that murine melanoma cells selected for high expression of α4, a counter-receptor for VCAM-1, demonstrated enhanced adhesion to lymphatic endothelial cells in vitro, and increased tumorigenicity and lymphatic metastasis in vivo, despite similar lymphatic vessel numbers. ^ Next, I examined the effects of growth factors that regulate lymphangiogenesis, and report that several growth factors are capable of activating survival and proliferation pathways of lymphatic endothelial cells. The dual protein tyrosine kinase inhibitor AEE788 (EGFR and VEGFR-2) inhibited the activation of Akt and MAPK in lymphatic endothelial cells responding to multiple growth factors. Moreover, oral treatment of mice with AEE788 decreased lymphatic vessel density and production of lymphatic metastasis by human colon cancer cells growing in the cecum of nude mice. ^ In the last set of experiments, I investigated the surgical management of lymphatic metastasis using a novel model of sentinel lymphadenectomy in live mice bearing subcutaneous B16-BL6 melanoma. The data demonstrate that this procedure when combined with wide excision of the primary melanoma, significantly enhanced survival of syngeneic C57BL/6 mice. ^ Collectively, these results indicate that the production of lymphatic metastasis depends on lymphangiogenesis, tumor cell adhesion to lymphatic endothelial cells, and proliferation of tumor cells in lymph nodes. Thus, lymphatic metastasis is a multi-step, complex, and active process that depends upon multiple interactions between tumor cells and tumor associated lymphatic endothelial cells. ^

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.^

Molecular consequences of the loss of 3'→5' exonuclease activity of DNA polymerases delta and epsilon

The DNA replication polymerases δ and ϵ have an inherent proofreading mechanism in the form of a 3'→5' exonuclease. Upon recognition of errant deoxynucleotide incorporation into DNA, the nascent primer terminus is partitioned to the exonuclease active site where the incorrectly paired nucleotide is excised before resumption of polymerization. The goal of this project was to identify the cellular and molecular consequences of an exonuclease deficiency. The proofreading capability of model system MEFs with EXOII mutations was abolished without altering polymerase function.^ It was hypothesized that 3'→5' exonucleases of polymerases δ and ϵ are critical for prevention of replication stress and important for sensitization to nucleoside analogs. To test this hypothesis, two aims were formulated: Determine the effect of the exonuclease active site mutation on replication related molecular signaling and identify the molecular consequences of an exonuclease deficiency when replication is challenged with nucleoside analogs.^ Via cell cycle studies it was determined that larger populations of exonuclease deficient cells are in the S-phase. There was an increase in levels of replication proteins, cell population growth and DNA synthesis capacity without alteration in cell cycle progression. These findings led to studies of proteins involved in checkpoint activation and DNA damage sensing. Finally, collective modifications at the level of DNA replication likely affect the strand integrity of DNA at the chromosomal level.^ Gemcitabine, a DNA directed nucleoside analog is a substrate of polymerases δ and ϵ and exploits replication to become incorporated into DNA. Though accumulation of gemcitabine triphosphate was similar in all cell types, incorporation into DNA and rates of DNA synthesis were increased in exonuclease defective cells and were not consistent with clonogenic survival. This led to molecular signaling investigations which demonstrated an increase in S-phase cells and activation of a DNA damage response upon gemcitabine treatment.^ Collectively, these data indicate that the loss of exonuclease results in a replication stress response that is likely required to employ other repair mechanisms to remove unexcised mismatches introduced into DNA during replication. When challenged with nucleoside analogs, this ongoing stress response coupled with repair serves as a resistance mechanism to cell death.^

Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase $\alpha$ are the molecular targets for two metal ions, Zn$\sp{2+}$ and Cd$\sp{2+},$ and an anticancer drug, F-ara-ATP.^ Human DNA ligases were purified to homogeneity and their AMP binding domains were mapped. Although their AMP-binding domains are similar, there could be difference between the two ligases in their DNA binding domains.^ The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP.^ A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex.^ F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3$\sp\prime$-terminus of DNA nick by DNA polymerase $\alpha.$^ All steps of the DNA ligation reaction were inhibited by Zn$\sp{2+}$ and Cd$\sp{2+}$ in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn$\sp{2+}$ and Cd$\sp{2+}$ showed their contradictory effects on the fidelity of the reaction by human DNA polymerase $\alpha.$ Zn$\sp{2+}$ decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd$\sp{2+}$ increased the frequencies of both misinsertion and mispair extension at very low concentration. Our data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported. ^

Molecular epidemiological studies on genetic predispositions to squamous cell carcinoma of the head and neck

Two molecular epidemiological studies were conducted to examine associations between genetic variation and risk of squamous cell carcinoma of the head and neck (SCCHN). In the first study, we hypothesized that genetic variation in p53 response elements (REs) may play roles in the etiology of SCCHN. We selected and genotyped five polymorphic p53 REs as well as a most frequently studied p53 codon 72 (Arg72Pro, rs1042522) polymorphism in 1,100 non-Hispanic White SCCHN patients and 1,122 age-and sex-matched cancer-free controls recruited at The University of Texas M. D. Anderson Cancer Center. In multivariate logistic regression analysis with adjustment for age, sex, smoking and drinking status, marital status and education level, we observed that the EOMES rs3806624 CC genotype had a significant effect of protection against SCCHN risk (adjusted odds ratio= 0.79, 95% confidence interval =0.64–0.98), compared with the -838TT+CT genotypes. Moreover, a significantly increased risk associated with the combined genotypes of p53 codon 72CC and EOMES -838TT+CT was observed, especially in the subgroup of non-oropharyneal cancer patients. The values of false-positive report probability were also calculated for significant findings. In the second study, we assessed the association between SCCHN risk and four potential regulatory single nucleotide polymorphisms (SNPs) of DEC1 (deleted in esophageal cancer 1) gene, a candidate tumor suppressor gene for esophageal cancer. After adjustment for age, sex, and smoking and drinking status, the variant -606CC (i.e., -249CC) homozygotes had a significantly reduced SCCHN risk (adjusted odds ratio = 0.71, 95% confidence interval = 0.52–0.99), compared with the -606TT homozygotes. Stratification analyses showed that a reduced risk associated with the -606CC genotype was more pronounced in subgroups of non-smokers, non-drinkers, younger subjects (defined as ≤ 57 years), carriers of TP53 Arg/Arg (rs1042522) genotype, patients with oropharyngeal cancer or late-stage SCCHN. Further in silico analysis revealed that the -249 T-to-C change led to a gain of a transcription factor binding site. Additional functional analysis showed that the -249T-to-C change significantly enhanced transcriptional activity of the DEC1 promoter and the DNA-protein binding activity. We conclude that the DEC1 promoter -249 T>C (rs2012775) polymorphism is functional, modulating susceptibility to SCCHN among non-Hispanic Whites. Additional large-scale, preferably population-based studies are needed to validate our findings.^

Apoptosis and the molecular complementation of bcl-2, myc and p53 in multistep lymphomagenesis

Follicular lymphoma is the most common lymphoid malignancy in humans. The bcl-2 transgenic mice, which mimic the human follicular lymphoma, initially exhibit a polyclonal hyperplasia due to the overriding of apoptosis by deregulated bcl-2. After a latency period of 15 month 20% of the animals developed clonal lymphomas. Approximately, 50% of these high grade lymphomas presented chromosomal translocations involving c-myc, suggesting that deregulation of this gene is important in the complementation with bcl-2. E$\mu$-myc x bcl-2 double transgenic mice were generated to assess the ability of this two genes to complement in an in vivo system. The double transgenic mice presented a shortened latency (3-4 weeks) and higher incidence of tumor development. Quantification of the extent of programmed cell death indicated that bcl-2 can abrogate the high rate of apoptotic cell death that results from myc deregulation. Bcl-2-Ig, E$\mu$-myc, and bcl-2/E$\mu$-myc lymphomas were examined using PCR-SSCP to detect the presence of p53 mutations in exons 5-9. A high incidence of p53 mutations in E$\mu$-myc lymphomas suggested that inactivating lesions of p53 may represent an important step in the genetic complementation of c-myc in lymphomagenesis. Surprisingly, p53 mutations were quite uncommon in bcl-2 lymphomas suggesting that inactivating mutations of p53 and overexpression of bcl-2 may not cooperate in lymphoma progression. To assess this question, we generated mice that contained a deregulated bcl-2 gene and were nullizygous for p53 (p53KO). No reduction in the tumor latency was observed in the p53KO/bcl-2-Ig hybrid mice when compared with p53 KO mice. Using splenic mononuclear cells isolated from p53KO mice and bcl-2 transgenic mice we demonstrated that bcl-2 suppresses p53 mediated apoptosis in response to DNA damage initiated by $\gamma$-radiation even though p53 protein is induced normally in the bcl-2 overexpressing cells. Western analysis of the expression of p53 target proteins after $\gamma$-radiation showed a correlation between the p53-dependent induction of bax protein after radiation and the ability of p53 to mediate apoptosis. ^

Biological effects of PEA3 expression in {\it HER-2\/}/{\it neu\/}-overexpressing human cancer cells and the molecular mechanisms of PEA3-mediated transcriptional repression on {\it HER-2\/}/{\it neu\/}

HER-2/neu is a receptor tyrosine kinase highly homologous with epidermal growth factor receptor. Overexpression and/or amplification of HER-2/neu has been implicated in the genesis of a number of human cancers, especially breast and ovarian cancers. Transcriptional upregulation has been shown to contribute significantly to the overexpression of this gene. Studies on the transcriptional regulation of HER-2/neu gene are important for understanding the mechanism of cell transformation and developing the therapeutic strategies to block HER-2/neu-mediated cancers. PEA3 is a DNA binding transcriptional factor and its consensus sequence exists on the HER-2/neu promoter. To examine the role of PEA3 in HER-2/neu expression and cell transformation, we transfected PEA3 into the human breast and ovarian cancer cells that overexpress HER-2/neu and showed that PEA3 dramatically represses HER-2/neu transcription. PEA3 suppresses the oncogenic neu-mediated transformation in mouse fibroblast NIH 3T3 cells. Expression of PEA3 selectively blocks the growth of human cancer cells that overexpress HER-2/neu and inhibits their colony formation. It does not occur in the cancer cells expressing basal level of HER-2/neu. Further studies in the orthotopic ovarian cancer model demonstrated that expression of PEA3 preferentially inhibits growth and tumor development of human cancer cells that overexpress HER-2/neu, the tumor-bearing mice survived significantly longer if treated by injection of the PEA3-liposome complex intraperitoneally. Immunoblotting and immunohistochemical analysis of the tumor tissues indicated that PEA3 mediates the tumor suppression activity through targeting HER-2/neu-p185. Thus, PEA3 is a negative regulator of HER-2/neu gene expression and functions as a tumor suppressor gene in the HER-2/neu-overexpressing human cancer cells.^ The molecular mechanisms of PEA3 mediated transcriptional repression were investigated. PEA3 binds specifically at the PEA3 site on HER-2/neu promoter and this promoter-binding is required for the PEA3 mediated transcriptional repression. Mutation of the PEA3 binding site on HER-2/neu promoter causes decreased transcriptional activity, indicating that the PEA3 binding site is an enhancer-like element in the HER-2/neu-overexpressing cells. We therefore hypothesized that in the HER-2/neu-overexpressing cells, PEA3 competes with a transactivator for binding to the PEA3 site, preventing the putative factor from activating the transcription of HER-2/neu. This hypothesis was supported by the data which demonstrate that PEA3 competes with another nuclear protein for binding to the HER-2/neu promoter in vitro, and expression of a truncated protein which encodes the DNA binding domain of PEA3 is sufficient to repress HER-2/neu transcription in the HER-2/neu-overexpressing human cancer cells. ^