Joan L. Cmarik

Activator protein 1 (AP-1)– and nuclear factor κB (NF-κB)–dependent transcriptional events in carcinogenesis

Abstract Generation of reactive oxygen species (ROS) during metabolic conversion of molecular oxygen imposes a constant threat to aerobic organisms. Other than the cytotoxic effects, many ROS and oxidants are also potent tumor promoters linking oxidative stress to carcinogenesis. Clonal variants of mouse epidermal JB6 cells originally identified for their differential susceptibility to tumor promoters also show differential reduction-oxidation (redox) responses providing a unique model to study oxidative events in tumor promotion. AP-1 and NF-κB, inducible by tumor promoters or oxidative stimuli, show differential protein levels or activation in response to tumor promoters in JB6 cells. We further demonstrated that AP-1 and NF-κB are both required for maintaining the transformed phenotypes where inhibition of either activity suppresses transformation response in JB6 cells as well as human keratinocytes and transgenic mouse. NF-κB proteins or extracellular signal–regulated kinase (ERK) but not AP-1 proteins are shown to be sufficient for conversion from transformation-resistant to transformation-susceptible phenotype. Insofar as oxidative events regulate AP-1 and NF-κB transactivation, these oxidative events can be important molecular targets for cancer prevention.

A novel transformation suppressor, Pdcd4, inhibits AP-1 transactivation but not NF-κB or ODC transactivation

Pdcd4 is a novel transformation suppressor that is highly expressed in promotion-resistant (P−) mouse epidermal JB6 cells but not in susceptible (P+) cells. Overexpression of pdcd4 cDNA in stably transfected P+ cells rendered cells resistant to tumor promoter-induced transformation, indicating that elevated expression of Pdcd4 protein is sufficient to suppress neoplastic transformation. To determine whether Pdcd4 suppresses neoplastic transformation through inhibiting known transformation required events, we examined the possibility that pdcd4 inhibited the activation of AP-1 or NF-κB dependent transcription or of ornithine decarboxylase (ODC) activity. Activation of AP-1-dependent transcriptional activity was inhibited by pdcd4 expression in a concentration dependent manner. In contrast, Pdcd4 slightly increased NF-κB-dependent transcription and did not alter ODC enzymatic activity. Previous studies suggested that activation of AP-1 was required for P+ cell transformation as well as for tumor promotion in vivo. These results indicate that Pdcd4 functions as a transformation suppressor, possibly through inhibiting AP-1 activation in combination with other factors such as enhancing NF-κB activation. Pdcd4 may thus constitute a useful molecular target for cancer prevention.

Tumor promoter induces high mobility group HMG-Y protein expression in transformation-sensitive but not -resistant cells

Elevated levels of high mobility group (HMG) nonhistone chromosomal proteins I and Y, alternatively spliced members of the HMG-I(Y) family of architectural transcription factors, have been linked with human cancer and with neo-plastic and metastatic phenotypes in model systems. To investigate whether HMG-I(Y) proteins may influence susceptibility to neoplastic transformation, HMG-I(Y) mRNA and protein levels were compared in the JB6 murine model of neoplastic progression. HMG-I(Y) mRNAs were expressed at very low levels in preneoplastic, transformation-resistant (P−) cell lines and were constitutively expressed at much higher levels in both transformation-sensitive (P+) and transformed (Tx) tumorigenic cell lines. HMG-I(Y) mRNAs were induced to higher levels by the tumor promoter 12-O-tetradecanoylphorbol acetate (TPA) and were sustained longer in P+ than in P− cells. Nevertheless, in both P− and P+ cells, primer extension analysis revealed that the same four major HMG-I(Y) gene transcription start sites were utilized with or without TPA treatment. RT–PCR revealed that there was always slightly more Y than I form mRNA present in all of the variant JB6 cell lines. Immunoblotting indicated that both HMG-I and -Y proteins increased in P+ cells in response to TPA treatment. Remarkably, in P− cells treated with TPA, only HMG-I (and not HMG-Y) protein levels increased. This unique differential TPA-induction of the HMG-Y protein in JB6 variants suggests a role for HMG-Y in mediating tumor promoter-induced neoplastic transformation. Furthermore, these results demonstrate that HMG-I and Y protein translation and/or stability is differently regulated in JB6 P− cells and provide the first indication that I and Y proteins may have different functions.

The Tyrosine Kinase sf-Stk and Its Downstream Signals Are Required for Maintenance of Friend Spleen Focus-Forming Virus-Induced Fibroblast Transformation

ABSTRACT Infection of erythroid progenitor cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia and eventually to erythroleukemia in susceptible strains of mice. The viral envelope protein, SFFV gp55, forms a complex with the erythropoietin receptor (EpoR) and a short form of the receptor tyrosine kinase Stk (sf-Stk), activating both and inducing Epo-independent proliferation. Recently, we discovered that coexpression of SFFV gp55 and sf-Stk is sufficient to transform NIH 3T3 and primary fibroblasts. In the current study, we demonstrate that sf-Stk and its downstream effectors are critical to this transformation. Unlike SFFV-derived erythroleukemia cells, which depend on PU.1 expression for maintenance of the transformed phenotype, SFFV gp55-sf-Stk-transformed fibroblasts are negative for PU.1. Underscoring the importance of sf-Stk to fibroblast transformation, knockdown of sf-Stk abolished the ability of these cells to form anchorage-independent colonies. Like SFFV-infected erythroid cells, SFFV gp55-sf-Stk-transformed fibroblasts express high levels of phosphorylated MEK, ERK, phosphatidylinositol 3-kinase (PI3K), Gab1/2, Akt, Jun kinase (JNK), and STAT3, but unlike virus-infected erythroid cells they fail to express phosphorylated STATs 1 and 5, which may require involvement of the EpoR. In addition, the p38 mitogen-activated protein kinase (MAPK) stress response is suppressed in the transformed fibroblasts. Inhibition of either JNK or the PI3K pathway decreases both monolayer proliferation and anchorage-independent growth of the transformed fibroblasts as does the putative kinase inhibitor luteolin, but inhibition of p38 MAPK has no effect. Our results indicate that sf-Stk is a molecular endpoint of transformation that could be targeted directly or with agents against its downstream effectors.

Neurodegeneration Induced by PVC-211 Murine Leukemia Virus Is Associated with Increased Levels of Vascular Endothelial Growth Factor and Macrophage Inflammatory Protein 1α and Is Inhibited by Blocking Activation of Microglia

ABSTRACT PVC-211 murine leukemia virus (MuLV) is a neuropathogenic retrovirus that has undergone genetic changes from its nonneuropathogenic parent, Friend MuLV, that allow it to efficiently infect rat brain capillary endothelial cells (BCEC). To clarify the mechanism by which PVC-211 MuLV expression in BCEC induces neurological disease, we examined virus-infected rats at various times during neurological disease progression for vascular and inflammatory changes. As early as 2 weeks after virus infection and before any marked appearance of spongiform neurodegeneration, we detected vessel leakage and an increase in size and number of vessels in the areas of the brain that eventually become diseased. Consistent with these findings, the amount of vascular endothelial growth factor (VEGF) increased in the brain as early as 1 to 2 weeks postinfection. Also detected at this early disease stage was an increased level of macrophage inflammatory protein 1α (MIP-1α), a cytokine involved in recruitment of microglia to the brain. This was followed at 3 weeks postinfection by a marked accumulation of activated microglia in the spongiform areas of the brain accompanied by an increase in tissue plasminogen activator, a product of microglia implicated in neurodegeneration. Pathological observations at the end stage of the disease included loss of neurons, decreased myelination, and mild muscle atrophy. Treatment of PVC-211 MuLV-infected rats with clodronate-containing liposomes, which specifically kill microglia, significantly blocked neurodegeneration. Together, these results suggest that PVC-211 MuLV infection of BCEC results in the production of VEGF and MIP-1α, leading to the vascular changes and microglial activation necessary to cause neurodegeneration.

Friend Spleen Focus-Forming Virus Activates the Tyrosine Kinase sf-Stk and the Transcription Factor PU.1 to Cause a Multi-Stage Erythroleukemia in Mice.

Hematological malignancies in humans typically involve two types of genetic changes: those that promote hematopoietic cell proliferation and survival (often the result of activation of tyrosine kinases) and those that impair hematopoietic cell differentiation (often the result of changes in transcription factors). The multi-stage erythroleukemia induced in mice by Friend spleen focus-forming virus (SFFV) is an excellent animal model for studying the molecular basis for both of these changes. Significant progress has been made in understanding the molecular basis for the multi-stage erythroleukemia induced by Friend SFFV. In the first stage of leukemia, the envelope protein encoded by SFFV interacts with and activates the erythropoietin (Epo) receptor and the receptor tyrosine kinase sf-Stk in erythroid cells, causing their Epo-independent proliferation, differentiation and survival. In the second stage, SFFV integration into the Sfpi1 locus activates the myeloid transcription factor PU.1, blocking erythroid cell differentiation, and in conjunction with the loss of p53 tumor suppressor activity, results in the outgrowth of malignant cells. In this review, we discuss the current level of understanding of how SFFV alters the growth and differentiation of erythroid cells and results in the development of erythroleukemia. Our knowledge of how SFFV causes erythroleukemia in mice may give us clues as to how the highly related human retrovirus XMRV causes malignancies in humans.

Use of Mouse JB6 Cells to Identify Molecular Targets and Novel Agents for Prevention of Carcinogenesis

Identification of the natural or synthetic agents that block the rate-limiting phase of tumor promotion is essential to the goal of cancer prevention. In vitro model systems to study the promotion process and to screen potential preventive agents continue to be important. One well established system representing preneoplastic to neoplastic progression is the JB6 murine epidermal model, which consists of promotion-resistant (P-) and promotion-sensitive (P+) preneoplastic cell lines as well as irreversibly transformed, tumorigenic cell lines. The model has been used to study the molecular mechanisms of tumor promotion, identify potential promoters and promotion inhibitors, elucidate differences between neoplastic and preneoplastic cells (e.g., identification of genes required for maintenance of tumor phenotype), and evaluate agents for reversion potential. Additionally, apoptosis response variants of one of the tumorigenic JB6 lines allow the study of programmed cell death, a process important to the prevention of or susceptibility to carcinogenesis. Exogenous promoters such as the phorbol esters and endogenous promoters such as epidermal growth factor, transforming growth factor, and tumor necrosis factor induce the anchorage-independent transformation of P+ cells. The antipromoting activity of a number of agents having cancer prevention potential has been demonstrated using P+ cells. These agents include classic antipromoters such as retinoic acid and food factors such as green or black tea and curcumin. Important insights into the mechanism of neoplastic transformation have been gained from comparison of JB6 P+ and P- cells, most notably the requirement for induction of transcription factor AP-1 transactivation activity. This understanding has led to the development of small molecule and transgene agents aimed at specifically blocking AP-1 activity; they are under evaluation for their ability to block cancer development.

Mechanism of action for the cytotoxic effects of the nitric oxide prodrug JS-K in murine erythroleukemia cells

The nitric oxide (NO) prodrug JS-K, a promising anti-cancer agent, consists of a diazeniumdiolate group necessary for the release of NO as well as an arylating ring. In this study, we research the mechanism by which JS-K kills a murine erythroleukemia cell line and determine the roles of NO and arylation in the process. Our studies indicate that JS-K inhibits the PI 3-kinase/Akt and MAP kinase pathways. This correlates with the activation of the tumor suppressor FoxO3a and increased expression of various caspases, leading to apoptosis. The arylating capability of JS-K appears to be sufficient for inducing these biological effects. Overall, these data suggest that JS-K kills tumor cells by arylating and inactivating signaling molecules that block the activation of a tumor suppressor.

Deregulation of Signal Transduction Pathways by Oncogenic Retroviruses

The proliferation and differentiation of cells is a highly regulated process that is controlled primarily at the level of the interaction of growth factors with their cell surface receptors. However, a variety of different retroviruses alter signal transduction pathways in infected cells, which is critical for the pathogenic process. Many studies have focused on how viral oncogenes and pathogenic genes associated with these retroviruses deregulate signal transduction pathways. Protein kinases, either encoded or activated by retroviruses, appear to play a major role. In this chapter, the effects of oncogenic retroviruses on signal transduction pathways will be reviewed for four oncogenic retroviruses: Abelson murine leukemia virus, S13 avian erythroblastosis virus, Friend spleen focus-forming virus, and Jaagsiekte sheep retrovirus. Each retrovirus uses a different mechanism to deregulate cell signaling.

Retrovirus-transformed erythroleukemia cells induce central nervous system failure in a new syngeneic mouse model of meningeal leukemia

Lack of suitable mouse models for central nervous system (CNS)-associated leukemias has hindered mechanism-guided development of therapeutics. By transplanting retrovirus-transformed mouse erythroleukemia cells into syngeneic mice, we developed a new animal model of meningeal leukemia associated with rapid paralysis. Necropsy revealed massive proliferation of the leukemic cells in the bone marrow (BM) followed by pathological angiogenesis and invasion of the leukemic cells into the meninges of the CNS. Further analysis demonstrated that the erythroleukemia cells secreted high levels of VEGF and preferentially adhered in vitro to fibronectin. This unique animal model for meningeal leukemia should facilitate studies of engraftment and proliferation of leukemic cells in the BM and their invasion of the CNS as well as pre-clinical evaluation of experimental therapeutics for CNS-associated leukemias.