The emergence of drug-resistance in cancer cells during chemotherapy remains a major obstacle in the treatment of neoplasia. Multidrug resistance (MDR) to a group of unrelated cytotoxic compounds can be conferred to eucaryotic cells by the expression of P-glycoprotein (Pgp), a putative plasma membrane transporter believed to mediate the efflux of these agents out of cells. A variety of agents are able to reverse this MDR phenotype by inhibiting the Pgp transporter. Blocking the action of this protein increases the effectiveness of cancer chemotherapeutic agents and, hence, has significant clinical implications. A mutant Pgp1 cDNA containing the substitution (Gly{dollar}\sp{lcub}338{rcub}{dollar}Ala{dollar}\sp{lcub}339{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}Pro{dollar}\sp{lcub}339{rcub}{dollar}) within the sixth transmembrane domain (tm6) has been cloned. The expression of this mutant confers an MDR phenotype preferentially resistant to actinomycin D. In this thesis we report that this MDR phenotype also has a decreased sensitivity toward reversal by cyclosporin A, while the sensitivity toward verapamil is unaltered. The accumulation of {dollar}\rm\lbrack\sp3H\rbrack{dollar} vincristine in cells expressing the wild-type Pgp1, not the mutant, increases dramatically in the presence of cyclosporin A, which correlates well with the reversal profile. We have altered only one amino acid residue at this location (Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar} or Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar}). The transfectants expressing the Pgp1 containing the proline substitution, rather than the alanine, demonstrate an MDR phenotype which is preferentially resistant to actinomycin D, and insensitive to reversal by cyclosporin A. Modeling the whole tm6 domain (with the Quanta modeling program and energy minimization by the CHARMm program) reveals that the proline substitution at position 339 rather than the alanine at 338 drastically changes the local {dollar}\alpha{dollar}-helice conformation, especially the polar side chain alignment along the hydrophilic side of this amphipathic {dollar}\alpha{dollar}-helice. We hypothesize that the Ala{dollar}\sp{lcub}339{rcub}{dollar} to Pro{dollar}\sp{lcub}339{rcub}{dollar} substitution, rather than the Gly{dollar}\sp{lcub}338{rcub}{dollar} to Ala{dollar}\sp{lcub}338{rcub}{dollar}, is the primary contributor to the aforementioned altered phenotype. We have also attempted to determine the functions of two spliced variants of Pgp1, previously cloned in this laboratory, by expressing them in an in vitro system. The biogenesis of one of the variants, ADX124, has also been investigated. We conclude that it is derived from a splicing event that involves the internal splicing signals that are maintained in the mature full-length Pgp1 transcript.

The development of a malignancy is a multistep process that is not clearly understood. Nonmalignant cells undergo a transformation process resulting in aberrantly proliferating malignant cells suggesting that the replication machinery of malignant cells is altered. In this study the DNA replication apparatus (the DNA synthesome) was examined to identify proteins altered in malignant cells. Analysis of malignant and nonmalignant cells by two dimensional gel electrophoresis (2D PAGE) demonstrated that many malignant cell types contain a unique, acidic form of proliferating cell nuclear antigen (PCNA). This protein is a 36 kD nuclear protein required for DNA replication and DNA repair. The unique form of PCNA was found in malignant breast, prostate, colon, cervical, brain and leukemia cell lines and in malignant human breast tumors and chronic myelogenous leukemia specimens. Serum collected from a breast cancer patient was analyzed and found to contain the cancer specific form of PCNA. Analysis of nonmalignant breast tissue and serum collected from cancer free individuals demonstrated that the cancer specific form of PCNA was not present. Further experiments were performed to characterize the unique form of PCNA. 2D PAGE analysis was performed on nonmalignant, transformed breast cell lines overexpressing the oncogenes c-myc (A1N4 myc) and SV40T (A1N4T). The cancer specific form of PCNA was present in these transformed cells. It was also determined that the acidic form of PCNA was not the result of growth stimulation or genetic mutation, suggesting that differential post-translational modification may be responsible. Although PCNA does undergo poly (ADP-ribosylation), 2D PAGE analysis demonstrated that the cancer specific form of PCNA was unmodified. The cancer specific form of PCNA appears to be a fundamental characteristic of malignancy and its role in tumorigenesis needs to be examined. These results suggest that epigenetic changes may contribute to the development of cancer.

Growth factor receptors of the tyrosine kinase subfamily I, ECFR/ErbB receptor family members, EGF receptor, erbB2, erbB3, and erbB4, mediate specific biological signals via heterodimerization among family members after ligand stimulation. The overall aim of this study was to examine erbB family member interaction mediating the biological effect of Heregulin (HRG), an erbB3 ligand. C-erbB2, an orphan receptor, was involved in the concentration-dependent pleiotropic responses of a breast cancer cell line to HRG, via heterodimerization with erbB3. HRG-mediated proliferation was maintained in erbB2-nonexpressing, antisense-erbB2 stable transfectants. In contrast, c-erbB2 was required for induction of HRC-mediated differentiation at high concentrations of HRG. To further study proteins that modulate the actions of erbB3, a receptor for HRG with an impaired tyrosine kinase activity, we isolated interacting proteins using a yeast two hybrid system screening. EBP1 and EBP2 have been identified as erbB3 interacting proteins using the yeast two hybrid system. EBP1 and EBP2 interacted with the juxtamembrane domain of erbB3 in the absence of ligand activation or intrinsic tyrosine kinase activity, in vitro. EBP1 and EBP2 mRNA was expressed in normal human epithelial tissues including brain, heart, and skeletal muscle, and in human carcinomas including breast, lung, and sarcoma. Overexpression of EBP1 or EBP2 suppressed colony growth and induced differentiation in a human breast carcinoma cell line, AU565. Treatment with HRG, but not with EGF, caused dissociation of EBP1 from erbB3 in vivo. EBP1 translocated from the cytoplasm into the nucleus following HRG stimulation. These findings suggest that HRG may regulate erbB3 function in part by directly linking receptor activation to nuclear activation through the translocation of an erbB3 interacting proteins. C-erbB2 was also partly involved in 17beta-estradiol-HRG induced cell growth inhibition of ER-negative erbB2-expressing breast carcinoma cell lines.

Murine polyomavirus is a DNA tumor virus that transforms cells in culture and induces tumors in mice. The major viral oncoproteins responsible for these phenomena are large T (LT) and middle T (MT) antigen. This thesis examines the ability of the T antigens to abrogate specific cell cycle check-points, which is a fundamental process in neoplastic transformation. We examined the ability of the T antigens to overcome the growth suppression function of p53. As a model system, a temperature sensitive p53 gene was introduced into mouse embryo fibroblasts from a p53 null mouse. At the permissive temperature (32C) a functionally wild-type p53 protein is expressed and arrests the growth of these cells in G1/G0 phase of the cell cycle. We introduced LT and MT antigen into these cells and demonstrated that LT overcame the growth arrest while MT did not. A LT mutant that was defective for binding the retinoblastoma tumor suppressor protein (pRB), was unable to overcome the growth arrest. This suggests that p53 directs the growth arrest through regulation of pRB and inactivation of pRB overcomes the arrest. Although MT did not overcome the arrest, mitogenic signals stimulated by MT were not inhibited by p53. We show that MT still associated with the signaling molecules PI3K and SHC, and increased the transcriptional activity of c-myc and AP1 in the p53 arrested cells. These results suggest that the growth arrest mediated by p53 is dominant over the proliferative signals generated by MT. Serum deprivation of NIH 3T3 cells results in growth arrest in G 0. Expression of MT in serum deprived cells was sufficient to reinitiate cell cycle progression. Following MT expression, levels of cyclin D and E increased and the level of p27 decreased resulting in increased G1 cyclin dependent kinase activity and hyperphosphorylation of pRB. These results indicate that MT abrogates the mitogen dependent check-point in G0. These studies demonstrate that LT overcomes the growth suppressive effects of p53 and MT substitutes for serum growth factors and overcomes growth arrest in G0. We conclude that the ability of the T antigens to overcome these distinct check-points is critical for cell transformation and presumably is also important for virus growth.

During carcinogenesis, the ability to control cell growth or death is lost. Transforming growth factor beta-1 (TGFbeta1) inhibits epithelial cell growth and plays a role in multistage carcinogenesis. To better understand TGFbeta1 in carcinogenesis, we have studied TGFbeta1 effects on cell shape and its mediators, which are critical to cell growth. We have also characterized a cell model appropriate for our TGFbeta 1 studies. The NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transfected NRK-52E cell lines, H/1.2 and H/6.1, were characterized. NRK-52E cells were differentiated, anchorage-dependent, non-tumorigenic, and contact-inhibited. H/1.2 and H/6.1 cells had increased growth rates, increased plasma membrane ruffling, altered cytoskeletal structure, anchorage-independent growth, and formed renal cell carcinoma-like tumors in vivo. H/6.1 cells formed more aggressive tumors in vivo than H/1.2, and were selected for further studies. Since Ras GTPases regulate cytoskeletal remodeling and we demonstrated H-ras oncogene-induced morphologic alterations, our cell model was validated for investigating the effects of TGFbeta1 on normal and tumorigenic cells. NRK-52E and H/6.1 cells had similar TGFbeta1 receptor expression and growth inhibition by TGFbeta1. NRK-52E treated with TGFbeta 1 showed cell flattening, actin protein expression, stress fiber formation, and increased vinculin fluorescence and expression. TGFbeta1 effects on NRK-52E cells induced talin fluorescence but not its expression. Biochemical analysis of F- and G-actin in NRK-52E cells showed significant TGFbeta1-induced increases. All of these effects were delayed, diminished, or absent in H1/6.1 cells. Since Rho GTPases are known to mediate stress fiber formation and TGFbeta1-induced transcription, we microinjected C3 transferase, a Rho inhibitor. C3 transferase depolymerized stress fibers in control cells but not in TGFbeta1-treated cells. Very high levels of C3 transferase eventually depolymerized all stress fibers in both treated and control cells. These results indicate that TGFbeta1 signaling in renal epithelial cells involves alterations in actin and focal adhesion proteins that differ between normal and H-ras-transformed cells. We suggest that TGFbeta1 modifies aspects of Rho GTPase signal transduction. Elucidating the mechanism by which TGFbeta1 induces cytoskeletal remodeling in tumor cells will reveal potential targets for cancer therapy.

A complete understanding of mammary cell differentiation is required in order to fully understand the molecular basis of breast cancer. Members of the ErbB receptor tyrosine kinase family have been implicated in the etiology and progression of many types of human cancers, including breast cancer. Heregulin (HRG), a ligand for the ErbB3 and ErbB4 receptors, plays a key role in mammary gland development. During pregnancy, HRG-mediated ErbB receptor signaling is responsible for the formation of lobuloalveoli followed by terminal mammary cell differentiation. In some mammary carcinoma cell lines, such as the AU565 cell line, HRG has been shown to induce cellular differentiation. In order to further characterize HRG-ErbB3 signaling, our laboratory previously used a yeast two-hybrid interaction screen to isolate ErbB3 binding proteins. EBP1(ErbB3 Binding Protein 1) was identified and has been demonstrated to dissociate from the ErbB3 receptor upon HRG stimulation. We further characterized this signaling protein by generating a polyclonal antibody against GST-EBP1. Protein and mRNA expression profiles were examined in a variety of cell lines. EBP1 expression was ubiquituous, and not limited to ErbB3-expressing cell lines. We also determined the biological effects of transfecting ebp1 cDNA into breast carcinoma cells. The EBP1 protein is basally phosphorylated in vivo on serine and threonine residues and this phosphorylation is enhanced after thirty minutes of heregulin stimulation. Both serine and threonine residues of GST-EBP1 fusion protein are phosphorylated by PKC in vitro. In vivo, we demonstrated that whereas HRG-induced phosphorylation of EBP1 occurs predominantly in a PKC-independent manner, basal EBP1 phosphorylation is dependent upon the activation of PKC. In conclusion, sustained activation of the ERK signaling pathway is essential for HRG-induced differentiation of the AU565 mammary carcinoma cell line. The ErbB3 binding phospho-protein EBP1 has also been implicated as a potential signaling molecule involved in this process. (Abstract shortened by UMI.)

Breast cancer is one of the most common malignant diseases in women. Several mechanisms have been proposed for the development and progression of breast cancer. Overexpression of growth factors or their receptors has been widely investigated as a potential pathway of breast malignancy. PCDGF is a novel growth factor characterized in our laboratory. There is significant evidence to suggest a role of PCDGF in human cancers. The purpose of this study was to investigate the role of PCDGF on tumorigenicity, estrogen dependence, endocrine therapy resistance and metastatic potential in human breast cancer. A model system to study the role of PCDGF and estrogen independence was developed by overexpressing PCDGF in human breast cancer MCF-7 cells and cultivating them in estrogen depleted condition. Results presented here show that PCDGF overexpression confers resistance to tamoxifen and fulvestrant in both in vitro and in vivo. PCDGF overexpression and estrogen depletion downregulate estrogen receptor alpha isoform, resulting in estrogen unresponsive cell growth. In addition, doxorubicin resistance was observed in PCDGF overexpressing cells. We found that PCDGF prevents apoptosis induced by tamoxifen, fulvestrant and doxorubicin. The key event in this process is that PCDGF maintains the upregulation of bcl-2 when treated with these agents. In addition, tamoxifen resistant cells express higher level of PCDGF. PCDGF transcriptionally activates estrogen inducible genes such as progesterone receptor and vascular endothelial growth factor (VEGF) via an ERa-dependent pathway. Tumors originated from PCDGF overexpressing cells express higher level of VEGF and angiopoietin-1. In addition, treatment with tamoxifen, in cooperation with PCDGF, stimulates VEGF expression in vitro and in vivo. This may explain why tamoxifen stimulates tumor growth from the PCDGF overexpressing cells but inhibits it in the wild-type cells. PCDGF induces cell migration through matrigel and stimulates matrix metalloprotease-9 secretion, suggesting an important role in metastasis. In summary, these studies provide a possible mechanism of antiestrogens and doxorubicin resistance in human breast cancer by overexpression of PCDGF. The results also show that PCDGF expression correlates with higher tumorigenicity and promotes angiogenesis and metastasis in human breast cancer.

In transformed cells the processing and translocation of lysosomal proteases are altered and protease inhibitors are deregulated. These alterations increase the capacity of neoplastic cells for degradation of extracellular matrix constituents and are thought to contribute to tumor invasion. I have tested the hypothesis that deregulation of intracellular protein degradation by lysosomes may also contribute to tumorigenesis by conferring a growth advantage to neoplastic cells, particularly in conditions of nutritional stress. In these conditions normal cells up-regulate the degradation of their cytoplasmic proteins to maintain amino acid and glucose homeostasis. To this end, I have studied protein turnover and the inducibility of lysosomal protein degradation in one mesenchymal (F9 12-1a) and three bronchial epithelial (BEAS-2B, a SV40 transformed clone of normal human bronchial cells (NHBE); BZR, a ras transfected tumorigenic clone of BEAS-2B; Calu-1, an epidermoid carcinoma cell line) transformed cells in culture. Protein degradation and synthesis were measured as the release of {dollar}\rm\sp{lcub}14{rcub}C{dollar}-valine from cells and incorporation of {dollar}\rm\sp{lcub}14{rcub}C{dollar}-valine into proteins. Autophagic degradation of cytoplasm was quantified by ultrastructural morphometry. I found that in F9 12-1a, BEAS-2B, and BZR cells basal proteolysis was down-regulated. In the transformed bronchial cells basal protein degradation was unaffected by cell density and did not appear to be regulated in parallel with rates of protein synthesis and cell proliferation. Prolonged nutritional deprivation up-regulated basal proteolysis in Calu-1 cells, this change was associated with the development of a more differentiated cellular phenotype. The induction of malignant phenotype did not affect basal proteolysis. Amino acid deprivation stimulated autophagic protein degradation equally well (2-fold) in transformed and normal cells. Serum growth factors did not regulate proteolysis in either normal or transformed bronchial cells. Induction of differentiated phenotype by {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar} or prolonged nutritional deprivation increased the inducibility of autophagic protein degradation in normal and transformed epithelial cells and upregulated proteolysis in response to deprivation of growth factors in Calu-1 cells. I conclude that transformation decreases the basal degradation of cytoplasmic proteins in some neoplastic cells. Down-regulation of proteolysis occurs independently of protein synthesis. Lysosomal proteolysis of transformed cells is not down-regulated. In normal and transformed cells proteolysis can be strikingly augmented by autophagy during acute or chronic nutritional stresses and can be further enhanced by inducing cell differentiation.

The anthracycline antibiotics are effective clinical anticancer agents for the treatment of leukemias, lymphomas, and solid tumors. The main biochemical effect of these drugs is believed to be interference with template DNA function during the transcription and replication processes. A highly potent anthracycline analog, cyanomorpholinodoxorubicin (CNM-DOX), was used to study the physicochemical effects of anthracyclines on duplex DNA. CNM-DOX complexed with DNA and increased the physicochemical stability of double-stranded DNA. The nature of this stabilized DNA-CNM-DOX complex is not DNA crosslinks. The interaction between CNM-DOX and DNA phosphate backbone, however, is crucial to highly stabilized CNM-DOX-DNA complex formation. Structure-activity relationship analysis revealed a link between the ability of anthracyclines to increase physicochemical stability of double-stranded DNA and their cytotoxicity. Since the enzyme helicase separates double-stranded DNA and prepares the strands for enzymatic replication or transcription, it is a potential cellular target for anthracyclines. We examined the effect of stabilized anthracycline-DNA complex on the dissociating activities of DNA helicases using a duplex DNA helicase substrate of {dollar}\rm\sp{lcub}32{rcub}P{dollar} labeled 17mer annealed to complementary M13 circular single stranded DNA. SV40 T-antigen helicase activity is potently inhibited by the anthracycline antibiotics with {dollar}\rm IC\sb{lcub}50{rcub}{dollar} values from 2 {dollar}\times{dollar} 10{dollar}\sp{lcub}-7{rcub}{dollar}M to 6 {dollar}\times{dollar} 10{dollar}\sp{lcub}-6{rcub}{dollar}M. Partially purified helicases from HeLa cells and murine FM3A cells also are potently inhibited by doxorubicin (DOX) with {dollar}\rm IC\sb{lcub}50{rcub}{dollar} values of 4 {dollar}\times{dollar} 10{dollar}\sp{lcub}-7{rcub}{dollar}M and 9 {dollar}\times{dollar} 10{dollar}\sp{lcub}-7{rcub}{dollar}M respectively. Both the enzymatic reaction rate and the maximal level of T-antigen helicase activity were significantly reduced with DOX treatment. While the DOX-DNA complex formation did not interfere with helicase associated ATPase activity, it blocked the conformational change of T-antigen during DNA dissociation process. These findings suggested that helicase is trapped by the DOX-DNA complex as a stable ternary complex (enzyme-DNA-drug), which subsequently prohibits the conformational change of helicase that is required for its DNA dissociating activity.