Matthew W. VanBrocklin

Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus

ABSTRACT We previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082–3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.

Antibody Microarray Profiling Reveals Individual and Combined Serum Proteins Associated with Pancreatic Cancer

We used antibody microarrays to probe the associations of multiple serum proteins with pancreatic cancer and to explore the use of combined measurements for sample classification. Serum samples from pancreatic cancer patients (n = 61), patients with benign pancreatic disease (n = 31), and healthy control subjects (n = 50) were probed in replicate experiment sets by two-color, rolling circle amplification on microarrays containing 92 antibodies and control proteins. The antibodies that had reproducibly different binding levels between the patient classes revealed different types of alterations, reflecting inflammation (high C-reactive protein, alpha-1-antitrypsin, and serum amyloid A), immune response (high IgA), leakage of cell breakdown products (low plasma gelsolin), and possibly altered vitamin K usage or glucose regulation (high protein-induced vitamin K antagonist-II). The accuracy of the most significant antibody microarray measurements was confirmed through immunoblot and antigen dilution experiments. A logistic-regression algorithm distinguished the cancer samples from the healthy control samples with a 90% and 93% sensitivity and a 90% and 94% specificity in duplicate experiment sets. The cancer samples were distinguished from the benign disease samples with a 95% and 92% sensitivity and an 88% and 74% specificity in duplicate experiment sets. The classification accuracies were significantly improved over those achieved using individual antibodies. This study furthered the development of antibody microarrays for molecular profiling, provided insights into the nature of serum-protein alterations in pancreatic cancer patients, and showed the potential of combined measurements to improve sample classification accuracy.

Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase

Lethal factor, the principal virulence factor of Bacillus anthracis, inhibits mitogen-activated protein kinase (MAPK) signaling by proteolytically cleaving MAPK kinases. Edema factor, another component of anthrax toxin, is an adenylate cyclase, which increases intracellular cAMP. Inhibition of MAPK signaling with either anthrax lethal toxin (LeTx) or small molecule MAPK kinase inhibitors triggers apoptosis in human melanoma cells. Normal melanocytes do not undergo apoptosis in response to MAPK inhibition but arrest in the G1 phase of the cell cycle. Importantly, in vivo treatment of human melanoma xenograft tumors in athymic nude mice with LeTx results in significant or complete tumor regression without apparent side effects, suggesting that inhibiting the MAPK signaling pathway may be a useful strategy for treating melanoma. Additionally, interrupting MAPK signaling with LeTx and elevating cAMP with anthrax edema toxin in both melanoma cells and melanocytes lead to dramatic melanin production, perhaps explaining the formation of blackened eschars in cutaneous anthrax.

Activated BRAF induces gliomas in mice when combined with Ink4a/Arf loss or Akt activation.

Mutations in receptor tyrosine kinase (RTK) growth factor receptors (epidermal growth factor receptor, platelet-derived growth factor receptor, MET and ERBB2), which result in downstream activation of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway and PI(3)K/Akt pathway, are found in almost all high-grade gliomas and MAPK signaling is necessary for continued glioma maintenance. In addition, BRAF is mutated in the majority of low-grade gliomas and its expression and activity is significantly increased in the majority of high-grade gliomas. Although the importance of RTKs and RAS signaling in glioma development has been shown, the role of BRAF has yet to be characterized. We evaluated the effect of activated BRAF in glioma formation using the retroviral replication-competent avian leukosis virus long terminal repeat, splice acceptor (RCAS)/TVA system to transfer genes encoding activated forms of BRAF, KRas, Akt and Cre to nestin-expressing neural progenitor cells in Ink4a/Arflox/lox mice in vivo. Although expression of activated BRAF alone is not sufficient for tumorigenesis, the combination of activated BRAF and Akt or BRAF with Ink4a/Arf loss is transforming. Interestingly, activated BRAF generates gliomas with characteristics similar to activated KRas in the context of Akt but not Ink4a/Arf loss. Our studies show a role for BRAF activation and signaling in glioma development and as potential target for glioma therapy.

Mitogen-Activated Protein Kinase Inhibition Induces Translocation of Bmf to Promote Apoptosis in Melanoma

Constitutive activation of the mitogen-activated protein kinase (MAPK) pathway is implicated in the development and progression of many human cancers, including melanoma. Mutually exclusive activating mutations in NRAS or BRAF have been identified in approximately 85% of melanomas, and components of this pathway have been developed as molecular targets for therapeutic intervention. We and others have shown that inhibition of this pathway with specific small molecule MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitors induces a wide range of apoptotic responsiveness in human melanoma cells both in vitro and in vivo. To define the molecular mechanism underlying variable apoptotic sensitivity of melanoma cells to MEK inhibition, we examined the expression and subcellular localization of Bcl-2 family members in a comprehensive set of human melanoma cell lines. Whereas the proapoptotic protein Bim was activated and localized to the mitochondrial membrane in all cell lines regardless of apoptotic sensitivity, Bmf activation and cytosolic translocation was exclusive to sensitive cells. In resistant cells, Bmf remained sequestered to the cytoskeleton through dynein light chain 2 (DLC2) binding. Overexpression of Bmf in resistant cells did not enhance apoptosis, whereas expression of mutant BmfA69P, which has decreased binding to DLC2, promoted cell death. Expression of BmfA69P mutants possessing the Bcl-2 homology 3 (BH3) domain mutation L138A, which impairs BH3 interactions, did not enhance apoptosis in resistant cells. RNA interference targeting Bim and Bmf provided protection from apoptosis induced by MEK inhibition. These results show a novel role for Bmf in promoting apoptosis and provide insight into the mechanism of apoptotic resistance to MEK inhibition in melanoma.

Differential oncogenic potential of activated RAS isoforms in melanocytes

RAS genes are mutated in approximately 30% of all human cancers. Interestingly, there exists a strong bias in favor of mutation of only one of the three major RAS genes in tumors of different cellular origins. NRAS mutations occur in approximately 20% of human melanomas, whereas HRAS and KRAS mutations are rare in this disease. To define the mechanism(s) responsible for this preference in melanocytes, we compared the transformation efficiencies of mutant NRAS and KRAS in immortal, non-transformed Ink4a/Arf-deficient melanocytes. NRAS mutation leads to increased cellular proliferation and is potently tumorigenic. In contrast, KRAS mutation does not enhance melanocyte proliferation and is only weakly tumorigenic on its own. Although both NRAS and KRAS activate mitogen-activated protein kinase signaling, only NRAS enhances MYC activity in these cells. Our data suggest that the activity of specific RAS isoforms is context-dependent and provide a possible explanation for the prevalence of NRAS mutations in melanoma. In addition, understanding this mechanism will have important implications for cancer therapies targeting RAS pathways.

AKT1 Activation Promotes Development of Melanoma Metastases

Metastases are the major cause of melanoma-related mortality. Previous studies implicating aberrant AKT signaling in human melanoma metastases led us to evaluate the effect of activated AKT1 expression in non-metastatic BRAF(V600E)/Cdkn2a(Null) mouse melanomas in vivo. Expression of activated AKT1 resulted in highly metastatic melanomas with lung and brain metastases in 67% and 17% of our mice, respectively. Silencing of PTEN in BRAF(V600E)/Cdkn2a(Null) melanomas cooperated with activated AKT1, resulting in decreased tumor latency and the development of lung and brain metastases in nearly 80% of tumor-bearing mice. These data demonstrate that AKT1 activation is sufficient to elicit lung and brain metastases in this context and reveal that activation of AKT1 is distinct from PTEN silencing in metastatic melanoma progression. These findings advance our knowledge of the mechanisms driving melanoma metastasis and may provide valuable insights for clinical management of this disease.

EFFICIENT LIPID-MEDIATED TRANSFECTION OF DNA INTO PRIMARY RAT HEPATOCYTES

Cationic lipids are an effective means for transfecting nucleic acids into a variety of cell types. Very few of these lipids, however, have been reported to be effective with primary cells. We report on the efficacy of several commercially available cationic lipid reagents to transfect plasmid DNA into primary rat hepatocytes in culture. The reagents tested in this study include TransfectAce, LipofectAmine, Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammoniumchloride (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate (DOTAP), and cetyltrimethyl-ammonium bromide/dioleoylphosphatidylethanol-amine (CTAB/DOPE). Electron micrographic (EM) studies indicate that similar size Lipofectin and DOTAP vesicles contain DNA-like material internally and that these vesicles attach to the cell membrane. DOTAP vesicles are multilamellar, appear as clusters, and have a high DNA-to-lipid ratio. Lipofectin vesicles appear to attach to the cell surface as individual vesicles. The EM observations are consistent with current theories on the mechanism of transfection by cationic lipids. While Lipofectin has proven to be effective in transfection studies of primary cells in culture, we have found DOTAP to be a viable alternative. DOTAP yields transfection rates in hepatocytes comparable to DOTMA and Lipofectin, however, at lower concentrations of reagent and at considerably less cost. Optimal conditions for transfecting 5 µg of plasmid DNA with DOTAP were achieved by utilizing multilamellar (vortexed) vesicles at a concentration of 15 µg DOTAP per 2 ml media in 60-mm plates for 2 h transfection time. In this study, DOTAP has proven to be economical, easy to prepare, and very effective in transfecting DNA into primary rat hepatocytes.

Activated MEK cooperates with Ink4a/Arf loss or Akt activation to induce gliomas in vivo

The RAS/RAF mitogen-activated protein kinase pathway (MAPK) is highly active in many tumor types including the majority of high-grade gliomas and expression of activated RAS or RAF in neural progenitor cells combined with either AKT activation or Ink4a/Arf loss leads to the development of high-grade gliomas in vivo. This strongly suggests that this pathway is necessary for glioma formation and maintenance. To further define the role of this pathway in the development of high-grade gliomas, we used the established RCAS/TVA glioma mouse model to test the ability of activated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK), a RAF effector, to induce tumors in vivo in the context of activated AKT or Ink4a/Arf loss. Although expression of activated MEK alone in neural progenitor cells is not sufficient for tumorigenesis, the combination of activated MEK and AKT or MEK with Ink4a/Arf loss is transforming. The data reveal that activation of the classical RAS/MAPK pathway, which is mediated through MEK, leads to the development of high-grade gliomas in vivo and suggest that MEK may be a relevant target for glioma therapy. To test this, we treated both mouse and human glioma cells with the MEK inhibitor PD0325901. Although this treatment induced apoptosis in a significant percentage of the cells, the effect was enhanced by combined treatment with the phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor NVP-BEZ235. Our results demonstrate that combined inhibition of MEK and PI3K/mTOR is a rational strategy for the treatment of high-grade gliomas and may be an effective adjuvant therapy for this disease.

Targeted delivery of NRASQ61R and Cre-recombinase to post-natal melanocytes induces melanoma in Ink4a/Arflox/lox mice.

We have developed a somatic cell gene delivery mouse model of melanoma that allows for the rapid validation of genetic alterations identified in this disease. A major advantage of this system is the ability to model the multi‐step process of carcinogenesis in immune‐competent mice without the generation and cross breeding of multiple strains. We have used this model to evaluate the role of RAS isoforms in melanoma initiation in the context of conditional Ink4a/Arf loss. Mice expressing the tumor virus A (TVA) receptor specifically in melanocytes under control of the dopachrome tautomerase (DCT) promoter were crossed to Ink4a/Arflox/lox mice and newborn DCT‐TVA/Ink4a/Arflox/lox mice were injected with retroviruses containing activated KRAS, NRAS and/or Cre‐recombinase. No mice injected with viruses containing KRAS and Cre or NRAS alone developed tumors; however, more than one‐third of DCT‐TVA/Ink4a/Arflox/lox mice injected with NRAS and Cre viruses developed melanoma and two‐thirds developed melanoma when NRAS and Cre expression was linked.