The interaction of P160 BCR with BCR-ABL gene products unique to Philadelphia chromosome leukemias

The BCR gene is involved in the pathogenesis of Philadelphia chromosome-positive (Ph$\sp1$) leukemias. Typically, the 5$\sp\prime$ portion of BCR on chromosome 22 becomes fused to a 5$\sp\prime$ truncated ABL gene from chromosome 9 resulting in a chimeric BCR-ABL gene. To investigate the role of the BCR gene product, a number of BCR peptide sequences were used to generate anti-BCR antibodies for detection of BCR and BCR-ABL proteins. Since both BCR and ABL proteins have kinase activity, the anti-BCR antibodies were tested for their ability to immunoprecipitate BCR and BCR-ABL proteins from cellular lysates by use of an immunokinase assay. Antisera directed towards the C-terminal portions of P160 BCR, sequences not present in BCR-ABL proteins, were capable of co-immunoprecipitating P210 BCR-ABL from the Ph$\sp1$- positive cell line K562. Re-immunoprecipitation studies following complete denaturation showed that C-terminal BCR antisera specifically recognized P160 BCR but not P210 BCR-ABL. These and other results indicated the presence of a P160 BCR/P210 BCR-ABL protein complex in K562 cells. Experiments performed with Ph$\sp1$-positive ALL cells and uncultured Ph$\sp1$-positive patient white blood cells established the general presence of BCR/BCR-ABL protein complexes in BCR-ABL expressing cells. However, two cell lines derived from Ph$\sp1$-positive patients lacked P160 BCR/P210 BCR-ABL complexes. Lysates from one of these cell lines mixed with lysates from a cell line that expresses only P160 BCR failed to generate BCR/BCR-ABL protein complexes in vitro indicating that P160 BCR and P210 BCR-ABL do not simply oligomerize.^ Two-dimensional tryptic maps were performed on both BCR and BCR-ABL proteins labeled in vitro with $\sp{32}$P. These maps indicate that the autophosphorylation sites in BCR-ABL proteins are primarily located within BCR exon 1 sequences in both P210 and P185 BCR-ABL, and that P160 BCR is phosphorylated in trans in similar sites by the activated ABL kinase of both BCR-ABL proteins. These results provide strong evidence that P160 BCR serves as a target for the BCR-ABL oncoprotein.^ K562 cells, induced to terminally differentiate with the tumor promoter TPA, show a loss of P210 BCR-ABL kinase activity 12-18 hours after addition of TPA. This loss coincides with the loss of activity in P160 BCR/P210 BCR-ABL complexes but not with the loss of the P210 BCR-ABL, suggesting the existence of an inactive form of P210 BCR-ABL. However, a degraded BCR-ABL protein served as the kinase active form preferentially sequestered within the remaining BCR/BCR-ABL protein complex.^ The results described in this thesis form the basis for a model for BCR-ABL induced leukemias which is presented and discussed. ^

Nuclear proteins interacting with an AP-1/CRE-like promoter sequence in the human TNF$\alpha$ gene

Monocyte developmental heterogeneity is reflected at the cellular level by differential activation competence, at the molecular level by differential regulation of gene expression. LPS activates monocytes to produce tumor necrosis factor-$\alpha$ (TNF). Events occurring at the molecular level necessary for TNF regulation have not been elucidated, but depend both on activation signals and the maturation state of the cell: Peripheral blood monocytes produce TNF upon LPS stimulation, but only within the first 72 hours of culture. Expression of c-fos is associated with monocytic differentiation and activation; the fos-associated protein, c-jun, is also expressed during monocyte activation. Increased cAMP levels are associated with down regulation of macrophage function, including LPS-induced TNF transcription. Due to these associations, we studied a region of the TNF promoter which resembles the binding sites for both AP-1(fos/jun) and CRE-binding protein (or ATF) in order to identify potential molecular markers defining activation competent populations of monocytic cells.^ Nuclear protein binding studies using extracts from THP-1 monocytic cells stimulated with LPS, which stimulates, or dexamethasone (Dex) or pentoxyfilline (PTX), which inhibit TNF production, respectively, suggest that a low mobility doublet complex may be involved in regulation through this promoter region. PTX or Dex increase binding of these complexes equivalently over untreated cells; approximately two hours after LPS induction, the upper complex is undetectable. The upper complex is composed of ATF2 (CRE-BP1); the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun-ATF2 complexes. The simultaneous presence of both complexes may reduce the amount of TNF transcription through competitive binding, while a loss of the upper (ATF2) and/or gain of the lower (jun-ATF2) allow increased transcription. AP-1 elements generally transduce signals involving PKC; the CRE mediates a cAMP response, involving PKA. Thus, this element has the potential of receiving signals through divergent signalling pathways. Our findings also suggest that cAMP-induced inhibition of macrophage functions may occur via down regulation of activation-associated genes through competitive binding of particular cAMP-responsive nuclear protein complexes. ^

Biologic and molecular analysis of tumor suppressor loci in human glioblastoma multiforme

Molecular and cytogenetic analyses of human glioblastomas have revealed frequent genetic alterations, including major deletions in chromosomes 9, 10, and 17, suggesting the presence of glioma-associated tumor suppressor genes on these chromosomes. To examine this hypothesis, copies of chromosomes 2, 4, and 10 derived from a human fibroblast cell line were independently introduced into a human glioma cell line, U251, by microcell-mediated chromosomal transfer. Successful transfer of chromosomes in each case was confirmed by resistance to the drug G418, indicating the presence of the neomycin-resistance gene previously integrated into each transferred chromosome. The presence of novel chromosomes and or chromosomal fragments was also demonstrated by molecular and karyotypic analyses. The hybrid clones containing either a novel chromosome 4 or chromosome 10 displayed suppression of the tumorigenic phenotype in vivo and suppression of the transformed phenotype in vitro, while cells containing a transferred chromosome 2 failed to alter their tumorigenic phenotype. The hybrid cells containing chromosome 4 or 10 exhibited a significant decrease in their saturation density, altered cellular morphology at high cell density, but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in growth of cells with chromosome 4 or 10 in soft agarose, with the number and size of the colonies being greatly reduced, compared to the parental or chromosome 2 containing cells. The introduction of chromosome 4 or 10 also completely suppressed tumor formation in nude mice. These studies indicate that chromosome 10, as hypothesized, and chromosome 4, a novel finding for gliomas, harbor tumor suppressor loci that may be directly involved in the initiation or progression of normal glial precursors to human glioblastoma multiforme. ^

Molecular analysis of TNF sensitivity in normal and Ha-{\it ras\/} transformed murine fibroblasts

Tumor necrosis factor (TNF) is known to have antiproliferative effects on a wide variety of tumor cells but proliferative effects on normal cells. However, the molecular basis for such differences in the action of TNF are unknown. The overall objectives of my research are to investigate the role of oncogenes in TNF sensitivity and delineate some of the molecular mechanisms involved in TNF sensitivity and resistance. To accomplish these objectives, I transfected TNF-resistant C3H mouse embryo fibroblasts (10T1/2) with an activated Ha-ras oncogene and determined whether these cells exhibit altered sensitivity to TNF. The results indicated that 10T1/2 cells transfected with an activated Ha-ras oncogene (10T-EJ) not only produced tumors in nude mice but also exhibited extreme sensitivity to cytolysis by TNF. In contrast, 10T1/2 cells transfected with the pSV2-neo gene alone were resistant to the cytotoxic effects of TNF. I also found that TNF-induced cell death was mediated through apoptosis. The differential sensitivity of 10T1/2 and 10T-EJ cell lines to TNF was not due to differences in the number of TNF receptors on their cell surface. In addition, TNF-resistant revertants isolated from Ha-ras-transformed, TNF-sensitive cells still expressed the same amount of p21 as TNF-sensitive cells and were still tumorigenic, suggesting that Ha-ras-induced transformation and TNF sensitivity may follow different pathways. Interestingly, TNF-resistant but not sensitive cells expressed higher levels of bcl-2, c-myc, and manganese superoxide dismutase (MnSOD) mRNA following exposure to TNF. However, TNF treatment resulted in a marginal induction of p53 mRNA in both TNF-sensitive and resistant cells. Based on these results I can conclude that (i) Ha-ras oncogene induces both transformation and TNF sensitivity, (ii) TNF-induced cytotoxicity involves apoptosis, and (iii) TNF-induced upregulation of bcl-2, c-myc, and MnSOD genes is associated with TNF resistance in C3H mouse embryo fibroblasts. ^

Immune responses against major histocompatibility complex and interleukin-2 gene transfected K1735 murine melanoma: Potential vaccines for the immunotherapy of cancer

Tumor specific immunity is mediated by cytotoxic T lymphocytes (CTL) that recognize peptide antigen (Ag) in the context of major histocompatibility complex (MHC) class I molecules and by helper T (Th) lymphocytes that recognize peptide Ag in the context of MHC class II molecules. The purpose of this study is (1) to induce or augment the immunogenicity of nonimmunogenic or weakly immunogenic tumors by genetic modification of tumor cells, and (2) to use these genetically altered cells in cancer immunotherapy. To study this, I transfected a highly tumorigenic murine melanoma cell line (K1735) that did not express constitutively either MHC class I or II molecules with syngeneic cloned MHC class I and/or class II genes, and then determined the tumorigenicity of transfected cells in normal C3H mice. K1735 transfectants expressing either $\rm K\sp{k}$ or $\rm A\sp{k}$ molecules alone produced tumors in normal C3H mice, whereas most transfectants that expressed both molecules were rejected in normal C3H mice but produced tumors in nude mice. The rejection of K1735 transfectants expressing $\rm K\sp{k}$ and $\rm A\sp{k}$ Ag in normal C3H mice required both $\rm CD4\sp+$ and $\rm CD8\sp+$ T cells. Interestingly, the $\rm A\sp{k}$ requirement can be substituted by IL-2 because transfection of $\rm K\sp{k}$-positive/A$\sp{\rm k}$-negative K1735 cells with the IL-2 gene also resulted in abrogation of tumorigenicity in normal C3H mice but not in nude mice. In addition, 1735 $(\rm I\sp+II\sp+)$ transfected cells can function as antigen presenting cells (APC) since they could process and present native hen egg lysozyme (HEL) to HEL specific T cell hybridomas. Furthermore, the transplantation immunity induced by K1735 transfectants expressing both $\rm K\sp{k}$ and $\rm A\sp{k}$ molecules completely cross-protected mice against challenge with $\rm K\sp{k}$-positive transfectants but weakly protected them against challenge with parental K1735 cells or $\rm A\sp{k}$-positive transfectants. Finally, I demonstrated that MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells can function as anti-cancer vaccines since they can abrogate the growth of established tumors and metastasis.^ In summary, my results indicate that expression of either MHC class I or II molecule alone is insufficient to cause the rejection of K1735 melanoma in syngeneic hosts and that both molecules are necessary. In addition, my data suggest that the failure of $\rm K\sp{k}$-positive K1735 cells to induce a primary tumor-rejection response in normal C3H mice may be due to their inability to induce the helper arm of the anti-tumor immune response. Finally, the ability of MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells to prevent growth of established tumors or metastasis suggests that these cell lines can serve as potential vaccines for the immunotherapy of cancer. (Abstract shortened by UMI.) ^

Gene therapy of cancer: Mechanisms of ``bystander effect'' killing in cells expressing the herpes simplex virus-thymidine kinase gene and its potential clinical applications

At the fore-front of cancer research, gene therapy offers the potential to either promote cell death or alter the behavior of tumor-cells. One example makes use of a toxic phenotype generated by the prodrug metabolizing gene, thymidine kinase (HSVtk) from the Herpes Simplex Virus. This gene confers selective toxicity to a relatively nontoxic prodrug, ganciclovir (GCV). Tumor cells transduced with the HSVtk gene are sensitive to 1-50 $\mu$M GCV; normal tissue is insensitive up to 150-250 $\mu$M GCV. Utilizing these different sensitivities, it is possible to selectively ablate tumor cells expressing this gene. Interestingly, if a HSVtk$\sp+$ expressing population is mixed with a HSVtk$\sp-$ population at high density, all the cells are killed after GCV administration. This phenomenon for killing all neighboring cells is termed the "bystander effect", which is well documented in HSVtk$\sp-$ GCV systems, though its exact mechanism of action is unclear.^ Using the mouse colon carcinoma cell line CT26, data are presented supporting possible mechanisms of "bystander effect" killing of neighboring CT26-tk$\sp-$cells. A major requirement for bystander killing is the prodrug GCV: as dead or dying CT26tk$\sp+$ cells have no toxic effect on neighboring cells in its absence. In vitro, it appears the bystander effect is due to transfer of toxic GCV-metabolites, through verapamil sensitive intracellular-junctions. Additionally, possible transfer of the HSVtk enzyme to bystander cells after GCV addition, may play a role in bystander killing. A nude mouse model suggests that in a 50/50 (tk$\sp+$/tk$\sp-$) mixture of CT26 cells the bystander eradication of tumors does not involve an immune component. Additionally in a possible clinical application, the "bystander effect" can be directly exploited to eradicate preexisting CT26 colon carcinomas in mice by intratumoral implantation of viable or lethally irradiated CT26tk$\sp+$ cells and subsequent GCV administration. Lastly, an application of this toxic phenotype gene to a clinical marking protocol utilizing a recombinant adenoviral vector carrying the bifunctional protein GAL-TEK to eradicate spontaneously-arisen or vaccine-induced fibrosarcomas in cats is demonstrated. ^

Ras signaling in tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF)-induced apoptosis is important in immunologic cytotoxicity, autoimmunity, sepsis, normal embryonic development, and wound healing. TNF exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. We found that enforced expression of an activated H-ras oncogene converted the non-tumorigenic TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells (10TEJ) that also became very sensitive to TNF-induced apoptosis. This finding suggested that the oncogenic form of H-Ras, in which the p21 is locked in the GTP-bound form, could play a role in TNF-induced apoptosis of these cells. To investigate whether Ras activation is an obligatory step in TNF-induced apoptosis, we introduced two different molecular antagonists of Ras, namely the Rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras transformed 10TEJ cells. Expression of either Rap1A or RasN17 in 10TEJ cells resulted in abrogation of TNF-induced apoptosis. Similar results were obtained by expression of either Ras antagonist in L929 cells, a fibroblast cell line that is sensitive to TNF-induced apoptosis but does not have a ras mutation. The effects of Rap-1A and RasN17 appear to be specific to TNF, since cytotoxicity induced by doxorubicin and thapsigargin are unaffected. Additionally, constitutive apoptosis sensitivity in isolated nuclei, as measured by activation of Ca$\sp{2+}$-dependent endogenous endonuclease, is not affected by Rap-1A or RasN17. Moreover, TNF treatment of L929 cells increased Ras-bound GTP, indicating that Ras activation is triggered by TNF. Thus, Ras activation is required for TNF-induced apoptosis in mouse cells. ^

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

Transcriptional regulation of the mouse $\alpha$2(I) collagen gene

The mouse $\alpha$2(I) collagen gene is specifically expressed in a limited number of cell types in the body including fibroblasts and osteoblasts. We had previously shown that a promoter containing the sequences between $-$350 and +54 bp was expressed at low levels in a cell- and tissue-specific fashion in transgenic mice. Further studies suggested that the sequence between $-$315 and $-$284 bp could mediate cell- and tissue-specific expression of reporter genes in cell culture and in transgenic mice. We report here characterization of the proteins binding to this segment and propose a model for the cell-specific expression conferred by this sequence. In this study we also identified a strong enhancer for the mouse $\alpha$2(I) collagen gene located approximately 13.5 to 19.5 kb upstream of the transcriptional start site. This enhancer segment is characterized by the presence of three cell-specific hypersensitive sites and can drive high levels of cell-specific expression of a heterologous 220-bp mouse $\alpha$1(I) collagen promoter. In the course of this study, we identified a novel zinc finger transcription factor (designated murine epithelial zinc finger, mEZF) which was transiently expressed in the mesenchymal cells which give rise to the skeletal primordia and the metanephric kidney during the early stages of embryogenesis. In newborn mice, the mEZF gene is expressed at high levels in differentiated epithelial cells of the skin, oral mucosa, tongue, esophagus, stomach and colon. Chromosomal mapping suggested that the mEZF gene mapped to mouse Chromosome 4 and that the human homolog of mEZF would likely map to human Chromosome 9q31. This region of the human genome contains tumor suppressor genes for basal cell carcinomas of the skin as well as for squamous cell carcinomas of various organs. We cloned and characterized the human homolog of mEZF and mapped its chromosomal position as a first step in determining whether or not this gene plays a role in the development of these tumors. ^

Human TNF receptor p75/80 gene structure and promoter characterization

Tumor necrosis factor receptor p75/80 ((TNF-R p75/80) is a 75 kDa type 1 transmembrane protein expressed predominately on cells of hematopoietic lineage. TNF-R p75/80 belongs to the TNF receptor superfamily characterized by cysteine-rich extracellular regions composed of three to six disulfide-linked domains. In the present report, we have characterized, for the first time, the complete gene structure for human TNF-R p75/80 which spans approximately 43 kbp. The gene consists of 10 exons (ranging from 34 bp to 2.5 kbp) and 9 introns (343 bp to 19 kbp). Consensus elements for transcription factors involved in T cell development and activation were noted in the 5$\sp\prime$ flanking region including TCF-1, Ikaros, AP-1, CK-2, IL-6RE, ISRE, GAS, NF-$\kappa$B and SP1, as well as an unusually high GC content and CpG frequency that appears characteristic of some TNF-R family members. The unusual (GATA)$\sb{\rm n}$ and (GAA)(GGA) repeats found within intron 1 may prove useful for further genome analysis within the 1p36 chromosomal locus. The human TNF-R p75/80 gene structure will permit further assessment of its involvement in normal hematopoietic cell development and function, autoimmune disease, and non-random translocations in hematopoietic malignancies. The region 1.8 kb 5$\sp\prime$ of the ATG was able to drive luciferase expression when transfected into cell lines expressing TNF-R p75/80. Further characterization of the 5$\sp\prime$-regulatory region will aid in determining factors and signal transduction pathways involved in regulating TNF-R p75/80 expression. ^

Targeting of {\it HER\/}-{\it 2\/}/{\it neu\/} with E1A gene therapy

Amplification or overexpression of HER-2/neu has been demonstrated in human cancers of the ovary, breast, lung and correlated with chemoresistance and poor clinic prognosis. We have previously found that the adenovirus type 5 early region 1A (E1A) gene product can repress the overexpression and suppress the tumorigenic potential of HER-2/neu-overexpressing cancer cells. In addition, E1A has been reported to induce apoptosis and inhibit the metastatic potential of tumor cells. Therefore, E1A could be considered as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. To develop an efficient HER-2/neu-targeting gene therapy with E1A, adenoviral vector or cationic liposome was used to introduce E1A into human ovarian, breast and lung cancer cells. Successful therapeutic effects were achieved.^ A replication-deficient adenovirus containing the E1A gene, Ad.E1A(+), was used to infect HER-2/neu-overexpressing human ovarian cancer cell line. Ovarian cancer growth in vitro and colony formation in soft agarose were greatly inhibited.^ To examine tumor suppressor function of E1A in breast cancer, we introduced E1A in vitro by adenovirus into both HER-2/neu-overexpressing and low-expressing human breast cancer cell lines. In HER-2/neu-overexpressing cells, E1A greatly inhibited tumor cell growth in vitro and colony formation in soft agarose. However, in low HER-2/neu expressing cancer cell lines, E1A could only reduce colony formation in soft agarose but had no significant effect on cell growth in monolayer, indicating different effects of E1A in these two types of cancer cells. To test the local therapeutic efficacy of E1A, we used either adenovirus- or liposome-mediated E1A gene delivery systems in an orthotopic breast cancer animal model.^ To test the therapeutic efficacy of systemically-delivered E1A in vivo lung cancer, we treated mice bearing intratracheal lung cancer by i.v. tail injections of Ad.E1A(+). As a result, Ad.E1A(+) suppressed HER-2/neu overexpression and inhibited intratracheal lung cancer growth. However, no significant tumor suppression effect of Ad.E1A(+) was observed in mice bearing HER-2/neu low expressing cell line when the same therapeutic procedure was followed. (Abstract shortened by UMI.) ^

New implications of E1A gene therapy in breast and ovarian cancer

A variety of human cancers overexpress the HER-2/neu proto-oncogene. Among patients with breast and ovarian cancers this HER-2/ neu overexpression indicates an unfavorable prognosis, with a shorter overall survival duration and a lower response rate to chemotherapeutic agents. Downregulation of HER-2/neu gene expression in cancer cells through attenuation of HER-2/neu promoter activity is, therefore, an attractive strategy for reversing the transformation phenotype and thus the chemoresistance induced by HER-2/neu overexpression. ^ A viral transcriptional regulator, the adenovirus type 5 E1A (early region 1A) that can repress the HER-2/neu promoter, had been identified in the laboratory of Dr. Mien-Chie Hung. Following the identification of the E1A gene, a series of studies revealed that repression of HER-2/neu by the E1A gene which can act therapeutically as a tumor suppressor gene for HER-2/ neu-overexpressing cancers. ^ The results of these preclinical studies became the basis for a phase I trial for E1A gene therapy among patients with HER-2/neu-overexpressing breast and ovarian cancer. In this dissertation, three primary questions concerned with new implications of E1A gene therapy are addressed: First, could E1A gene therapy be incorporated with conventional chemotherapy? Second, could the E1A gene be delivered systemically to exert an anti-tumor effect? And third, what is the activity of the E1A gene in low-HER-2/neu-expressing cancer cells? ^ With regard to the first question, the studies reported in this dissertation have shown that the sensitivity of HER-2/neu-overexpressing breast and ovarian cancer to paclitaxel is in fact enhanced by the downregulation of HER-2/neu overexpression by E1A. With regard to the second question, studies have shown that the E1A gene can exert anti-tumor activity by i.v. injection of the E1A gene complexed with the novel cationic liposome/protamine sulfate/DNA type I (LPDI). And with regard to the third question, the studies of low-HER-2/ neu-expressing breast and ovarian cancers reported here have shown that the E1A gene does in fact suppress metastatic capability. It did not, however, suppress the tumorigenicity. ^ Three conclusions can be drawn from the experimental findings reported in this dissertation. Combining paclitaxel with E1A gene therapy may expand the implications of the gene therapy in the future phase II clinical trial. Anti-tumor activity at a distant site may be achieved with the i.v. injection of the E1A gene. Lastly when administered therapeutically the anti-metastatic effect of the E1A gene in low-HER-2/neu-expressing breast cancer cells may prevent metastasis in primary breast cancer. (Abstract shortened by UMI.)^

Relationships between the alterations of tumor suppressor genes, risk factors and clinical outcomes of gliomas

The relationship between MMAC/PTEN, DMBT1 and the progression and prognosis of glioma, and the association between the alterations of MMAC/PTEN, p53, p16, and Rb and some cancer risk factors, such as smoking, exposure to radiation, family cancer history, and previous cancer history, were assessed in 4 studies. ^ By allelic deletion analysis, MMAC/PTEN locus was shown to be frequently lost in glioblastomas multiforme (GM) but maintained in most lower-grade astrocytic tumors. DMBT1 locus, however, was frequently lost in all grades of gliomas examined. The potential biological significance of these two regions was frontier assessed by examining microcell-hybrids that contained various fragments of 10q. Somatic cell hybrid clones that retained the MMAC/PTEN locus have less transformed phenotypes, exhibiting an inability to grow in soft agarose. On the other hand, the presence or absence of DAMT1 did not correlate with any in vitro phenotype assessed in our model system. Further, Cox proportional hazards regression analysis, adjusted for age at surgery and histologic grades (GM, and non-GM), showed that without LOH at the MMAC/PTEN locus had a significantly better prognosis than did patients with LOH at MMAC/ PTEN (hazard ratio = 0.5; 95% Cl = 0.28–0.89; P = 0.018). Furthermore, status of LOH at MMAC/PTEN was found to be significantly associated with age, while that for DMBT1 was not. These results suggest that the DMBT1 may be involved early in the oncogenesis of gliomas, while alterations in the MMAC /PTEN may be a late event in the oncogenesis related with progression of gliomas and provide a significant prognostic marker for patient survival. ^ The associations between 4 cancer risk factors and 4 tumor suppressor genes were assessed. The expression of p16 was observed to be associated with current smoking (adjusted OR = 1.9, 95% CI = 1.02–3.6) but not the former smoking (adjusted OR = 1.1, 95% Cl = 0.5–3.5). The expression of p53 was found to be associated with the family cancer history (OR = 3.5, 95% Cl = 1.07–11 for patients with first-degree family history of cancer). MMAC/ PTEN was associated with the histologic grade (OR = 2.8, 95% CI = 1.2–6.6) and age (P = 0.035). Also, the OR for LOH around MMAC/PTEN in patients with a family history of cancer was elevated (OR = 1.9, 95% CI = 0.8–4.6 for patients with first-degree family history of cancer). The associations between exposure and the alterations of tumor suppressor genes, between smoking and p16, between family history of cancer and p53 and MMAC/PTEN, provide suggestive evidences that those exposures are related to the development of gliomas. ^

COMPARISON OF DNA TRANSFECTION AND MICROCELL-MEDIATED CHROMOSOME TRANSFER FOR DETECTING TRANSFORMING GENES IN HUMAN TUMOR CELL LINES

DNA mediated gene transfection is an important tool for moving and isolating genes from one cell type and putting them into a foreign genetic background. DNA transfection studies have been done routinely in many laboratories to identify and isolate transforming sequences in human tumors and tumor cell lines. A second technique, microcell-mediated chromosome transfer, allows the transfer of small numbers of intact human chromosome from one cell to another. This work was done to compare the efficiency of these two techniques in the transformation of NIH 3T3 mouse fibroblast cells.^ My intent in comparing these two techniques was to see if there was a difference in the transforming capability of DNA which has been purified of all associated protein and RNAs, and that of DNA which is introduced into a cell in its native form, the chromosome. If chromosomal sequences were capable of transforming the 3T3 cells in culture, the method could then be used as a way to isolate the relevant tumorigenic chromosomes from human tumors.^ The study shows, however, that even for those cell lines that contain transforming sequences identified by DNA-mediated gene transfer, those same sequences were unable to transform 3T3 cells when introduced to the cells by somatic fusion of human tumor microcells. I believe that the human transforming sequences in their original genetic conformation are not recognized by the mouse cell as genes which should be expressed; therefore, no noticeable transformation event was selected by this technique. ^

GENETIC EVENTS PREDISPOSING TO WILMS' TUMOR

Wilms' tumor (WT) is a childhood embryonic tumor of the kidney. In some cases, WT has been associated with a chromosome deletion in the region 11p13. The majority of WT cases, however, have normal karyotypes with no discernable deletions or rearrangements of chromosome 11.^ To study the genetic events predisposing to the development of WT, I have used a number of gene markers specific for chromosome 11. Gene probes for human catalase and apolipoprotein A1 were localized to chromosome 11 by in situ hybridization. A number of other probes previously mapped to chromosome 11 were also used. Nine WT patients who were heterozygous for at least one 11p marker were shown to lose heterozygosity in their tumor DNA. Gene dosage experiments demonstrated that two chromosomes 11 were present although loss of heterozygosity had occurred in all but two cases. By using gene probes from the short and long arms of chromosome 11, I discerned that loss of heterozygosity was due to somatic recombination in four cases, chromosome deletion in two cases, and chromosome loss and reduplication or somatic recombination in these cases. Examination of DNAs from the parents of six of these patients indicated that the alleles that were lost in tumor tissues were alleles inherited from the mother. In sporadic WT cases one would expect the loss of alleles to be random. These data suggest that the loss of alleles resulting in the development of WT is not a random event, however, the significance of this is not known. ^