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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.

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.

The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells.

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.

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.

A novel protein kinase, polyploidy-associated protein kinase (PAPK), was isolated using a subtraction cDNA library approach from a mouse erythroleukemia cell line that had been induced to polyploidy after serum withdrawal. PAPK shares homology with members of the Ste20/germinal center kinase family of protein kinases and is ubiquitously expressed as two spliced forms, PAPK-A and PAPK-B, that encode for proteins of 418 and 189 amino acids, respectively. The expression of endogenous PAPK-A protein increased after growth factor withdrawal in murine hematopoietic and fibroblast cells. When tested in an in vitro kinase assay, PAPK-A was activated in response to the stress-inducing agent hydrogen peroxide and slightly by fetal calf serum. Biochemical characterization of the PAPK-A-initiated pathway revealed that this novel kinase does not affect MAP kinase activity but can stimulate both c-Jun N-terminal kinase 1 (JNK1) and ERK6/p38 gamma. The kinase activity of PAPK appears to be required for the activation of ERK6/p38 gamma but not JNK1. When an inducible construct of PAPK-A was expressed in stably transfected NIH3T3 cells, the cells exhibited distinct cytoskeletal changes and became resistant to apoptotic cell death induced by serum withdrawal, effects of PAPK that require its kinase activity. These data suggest that PAPK is a new member of the Ste20/germinal center kinase family that modulates cytoskeletal organization and cell survival.

Changes that occur during tumor promotion, the rate-limiting phase of multistep carcinogenesis, may offer the best targets for prevention of cancer or reversal of early disease. The murine epidermal JB6 promotion-sensitive (P+) and -resistant (P-) cell lines provide a cell culture model for tumor promoter-induced neoplastic transformation ideally suited to the identification of molecular events that mediate or inhibit transformation. A differential display comparison of P+ and P- cell mRNAs yielded seven differentially expressed sequences. One of the sequences preferentially expressed in P- cells identified an approximately 3. 6-kb message that was induced to higher levels in P- cells following exposure to the tumor promoter 12-O-tetradecanoylphorbol acetate than in P+ cells. The message was detected in mRNA from heart, lung, and spleen. cDNA cloning of the P- preferential sequence revealed a high degree of identity to human pleckstrin (PLEK), the major PKC substrate in platelets (Tyers et al., 1988, Nature 333: 470). We report the complete mouse cDNA sequence of pleckstrin and the localization of the gene to chromosome 11, its expression in a nonhematopoetic cell line, and its potential role in blocking neoplastic transformation.

An mRNA differential display comparison of mouse JB6 promotion-sensitive (P+) and -resistant (P−) cells identified a novel gene product that inhibits neoplastic transformation. The JB6 P+ and P− cells are genetic variants that differ in their transformation response to tumor promoters; P+ cells form anchorage-independent colonies that are tumorigenic, and P− cells do not. A differentially displayed fragment,A7-1, was preferentially expressed in P− cells at levels ≥10-fold those in P+ cells, making its mRNA a candidate inhibitor of neoplastic transformation. AnA7-1cDNA was isolated that was identical to murinePdcd4gene cDNAs, also known asMA-3orTIS, and analogous to humanH731and197/15a. Until now, the function of the Pdcd4 protein has been unknown. Paralleling the mRNA levels, Pdcd4 protein levels were greater in P− than in P+ cells.Pdcd4mRNA was also expressed at greater levels in the less progressed keratinocytes of another mouse skin neoplastic progression series. To test the hypothesis that Pdcd4 inhibits tumor promoter-induced transformation, stable cell lines expressing antisensePdcd4were generated from parental P− cells. The reduction of Pdcd4 proteins in antisense lines was accompanied by acquisition of a transformation-sensitive (P+) phenotype. The antisense-transfected cells were reverted to their initial P− phenotype by overexpression of aPdcd4sense fragment. These observations demonstrate that the Pdcd4 protein inhibits neoplastic transformation.

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 JB6 mouse epidermal cell system has been extensively used as an in vitro model for the study of tumor promotion. The present study aimed to assess the relevance of monolayer measurements to the process of transformation, which is induced more efficiently under anchorage-independent (AI) conditions. Although it would be ideal to use identical conditions for studying tumor promoter-induced transformation and biochemical and molecular events that may cause the process, it is not feasible in the case of soft agar conditions because cells cannot be readily recovered. In the present report, we used liquid medium over agar as an AI condition that permitted efficient recovery of cells. Responses to tumor promoter have been compared with those in monolayer and semisolid agar. Results indicate that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced similar magnitude concentration-dependent transformation of JB6 cells under both of the AI conditions, namely soft agar and over-agar. Under anchorage-dependent (AD) conditions of exposure to TPA, the transformation efficiency was much lower than that seen under AI conditions. Mechanical detachment of monolayer cells after 5-10 days TPA exposure enriched the transformed phenotype. Activator protein 1 transcriptional activity measured at 12 h was induced equally under AD and AI conditions, and thus is not an early limiting event that could explain the lower transformation efficiency seen under AD conditions. To summarize, the over-agar and monolayer assays described in this study can be considered valid for the study of early biochemical and molecular events relevant to the promotion of transformation measured in soft agar.

We recently reported that infection of rats with the neurodegenerative disease-causing retrovirus PVC-211 MuLV results in elevated levels of the chemokine MIP-1α followed by the accumulation of activated microglia in the brain. To investigate the importance of MIP-1α in recruitment of microglia to the brain, we treated rats with MIP-1α antibodies before and after PVC-211 MuLV infection. This caused a delay in the development of paralysis which was associated with a decrease in activated microglia without affecting virus expression. To determine the source of activated microglia, rats were splenectomized 4days after virus infection. Splenectomized rats showed a delay in disease development that was associated with decreased numbers of activated microglia without affecting virus expression. Together, these results suggest that MIP-1α is directly involved in the neurodegeneration induced in rats by PVC-211 MuLV by recruiting macrophages/microglia from the periphery into regions of the brain that eventually become diseased.

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.

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.

Most molecular biology and biochemical analyses use cultured cells grown in anchorage-dependent monolayer conditions. The standard oncogenic transformation assay for cell lines is usually performed in soft agar rather than in monolayers because of the higher transformation efficiency of cells in soft agar. However, cells suspended in soft agar cannot be readily recovered for studying inducible biochemical and molecular events. We developed an over-agar assay that enables us to study tumor promoter-induced cell transformation and the associated biochemical or molecular events under anchorage-independent conditions.

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-kappaB 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-kappaB-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-kappaB activation. Pdcd4 may thus constitute a useful molecular target for cancer prevention.

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.

The 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) genes are a set of primary response genes induced in Swiss 3T3 cells by TPA. They include three transcription factors, a prostaglandin synthase, and three proteins of unknown function. To ascertain which, if any TIS genes might be involved in tumor promotion, we examined the expression of these genes in response to tumor promoters in transformation promotion-sensitive (P+) and -resistant (P-) JB6 murine epidermal cells, a model used to identify events relevant to promotion. A subset of TIS genes (TIS1,TIS21), TIS10, and TIS21) was preferentially induced by TPA in P-cells. In addition, TIS1 and TIS21 mRNAs were preferentially induced in P-cells by epidermal growth factor, another transformation promoter that distinguishes P+ from P- cells. TIS1 and TIS21 protein levels were also greater in TPA-treated P- cells than P+ cells. Forskolin, a cAMP-elevating anti-promoter, increased TPA-induced levels of TIS1, TIS10, and TIS21 mRNAs in P+ cells, ruling in potential roles for these genes in modulating tumor promotion. The anti-promoters fluocinolone acetonide, retinoic acid, and superoxide dismutase did not enhance TPA-induced levels of TIS1 and TIS21 mRNAs in P+ cells, suggesting that these inhibitors may act on other promotion-relevant genes. TIS1 encodes a member of the steroid receptor superfamily. TIS21 encodes a protein of unknown function with strong sequence similarity to BTG1, a proposed “anti-proliferative gene” (Rouault JP, Rimokh R, Tessa C, et al., EMBO J 11:1663–1670, 1992). Preferential induction by multiple promoters of these TIS genes in P- cells and enhancement of their induction in P+ cells by the anti-promoter forskolin make TIS1 and TIS21 candidates for promotion suppressor genes. ©1994 Wiley-Liss, Inc.

Experiments were conducted with male and female rats (12 per group) dosed by gavage with 2 or 20 mg (based on the free amine) doxylamine succinate containing about 10 microCi 14C-doxylamine succinate to determine distribution and excretion of the activity as a function of dose and sex with time. Urine and feces were collected at intervals up to 72 hr. Most of the dose (approximately equal to 70%) was eliminated in the first 24 hr after dosing and 95 to 100% of the dose was recovered during the 72-hr course of the experiments with both sexes and dose levels. Less than 1% of the total dose remained in the rats at the end of the test period. The urinary route of elimination was more predominant than the fecal route in both sexes given the 20-mg dose. The fecal route predominates in low-dose males whereas there is no significant difference between urinary and fecal routes of elimination in low-dose females. Preliminary characterization of urinary metabolite form using extraction techniques shows 99% of the metabolites to be in the polar conjugated form.