Treatment of a human papillomavirus type 31b-positive cell line with benzo[a]pyrene increases viral titer through activation of the Erk1/2 signaling pathway.

Numerous epidemiological studies have implicated cigarette smoking as a cofactor in the progression to cervical cancer. Tobacco-associated hydrocarbons have been found in cervical mucus, suggesting a possible interaction with human papillomavirus (HPV)-infected cells. The polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) is a major component of cigarette smoke condensate that has received significant attention due to its ability to induce carcinogenesis. We have previously demonstrated by conventional methods for determining viral titer that high concentrations of BaP increase HPV31b titers within the context of organotypic raft cultures compared with the level for vehicle controls. However, a definitive mechanism for explaining this increase in viral titer was lacking. Here, we show that BaP treatment activates the Ras-Raf-Mek1/2-Erk1/2 signaling pathway. The importance of Erk1/2 pathway activation to the BaP-mediated increase in viral titer was determined by Erk pathway inhibition with multiple Erk1/2 pathway inhibitors. Finally, BaP treatment activated p90RSK and its downstream target CDK1. These data indicate that the Erk1/2 signaling pathway plays an important role in mediating the response to BaP treatment that ultimately leads to increased viral titers.

Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells.

BACKGROUND: In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery. RESULTS: AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. CONCLUSION: AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.

Downregulation of Cdc2/CDK1 kinase activity induces the synthesis of noninfectious human papillomavirus type 31b virions in organotypic tissues exposed to benzo[a]pyrene.

Epidemiological studies suggest that human papillomavirus (HPV)-infected women who smoke face an increased risk for developing cervical cancer. We have previously reported that exposure of HPV-positive organotypic cultures to benzo[a]pyrene (BaP), a major carcinogen in cigarette smoke, resulted in enhanced viral titers. Since BaP is known to deregulate multiple pathways of cellular proliferation, enhanced virion synthesis could result from carcinogen/host cell interaction. Here, we report that BaP-mediated upregulation of virus synthesis is correlated to an altered balance between cell cycle-specific cyclin-dependent kinase (CDK) activity profile compared with controls. Specifically, BaP treatment increased accumulation of hyperphosphorylated retinoblastoma protein (pRb) which coincided with increased cdc2/CDK1 kinase activity, but which further conflicted with the simultaneous upregulation of CDK inhibitors p16(INK4) and p27(KIP1), which normally mediate pRb hypophosphorylation. In contrast, p21(WAF1) and p53 levels remained unchanged. Under these conditions, CDK6 and CDK2 kinase activities were decreased, whereas CDK4 kinase activity remained unchanged. The addition of purvalanol A, a specific inhibitor of CDK1 kinase, to BaP-treated cultures, resulted in the production of noninfectious HPV type 31b (HPV31b) particles. In contrast, infectivity of control virus was unaffected by purvalanol A treatment. BaP targeting of CDK1 occurred independently of HPV status, since BaP treatment also increased CDK1 activity in tissues derived from primary keratinocytes. Our data indicate that HPV31b virions synthesized in the presence of BaP were dependent on BaP-mediated alteration in CDK1 kinase activity for maintaining their infectivity.

Adeno-associated virus type 2 infection of nude mouse human breast cancer xenograft induces necrotic death and inhibits tumor growth.

We have previously reported that infection with the non-pathogenic, tumor suppressive, wild-type adeno-associated virus type 2 (AAV2) inhibited proliferation of breast cancer-derived lines representing both weakly invasive (MCF-7 and MDA-MB-468), as well as aggressive (MDA-MB-231) cancer types. AAV2-induced death occurred via targeting pathways of apoptosis and necrosis. In contrast, normal human mammary epithelial cells were unaffected upon AAV2 infection. The current study characterizes AAV2 infection and subsequent death of the highly aggressive, triple-negative (ER(-)/PR(-)/HER2(-)) MDA-MB-435 cell line derived from metastatic human breast carcinoma. Monolayer MDA-MB-435 cultures infected with AAV2 underwent complete apoptotic cell death characterized by activation of caspases -7, -8, and -9 and PARP cleavage. Death was further correlated with active AAV2 genome replication and differential expression of viral non-structural proteins Rep78 and Rep52. Cell death coincided with increased entry into S and G 2 phases, upregulated expression of the proliferation markers Ki-67 and the monomeric form of c-Myc. Expression of the p16(INK4), p27(KIP1), p21(WAF1), and p53 tumor suppressors was downregulated, indicating marked S phase progression, but sharply contrasted with hypo-phosphorylated pRb. In parallel, MDA-MB-435 breast tumor xenografts which received intratumoral injections of AAV2 were growth retarded, displayed extensive areas of necrosis, and stained positively for c-Myc as well as cleaved caspase-8. Therefore, AAV2 induced death of MDA-MB-435 xenografts was modulated through activation of caspase-regulated death pathways in relation to signals for cell cycle controls. Our findings provide foundational studies for development of novel AAV2 based therapeutics for treating aggressive, triple-negative breast cancer types.

Human papillomavirus type 18 chimeras containing the L2/L1 capsid genes from evolutionarily diverse papillomavirus types generate infectious virus.

Papillomaviruses (PVs) comprise a large family of viruses infecting nearly all vertebrate species, with more than 100 human PVs identified. Our previous studies showed that a mutant chimera HPV18/16 genome, consisting of the upper regulatory region and early ORFs of HPV18 and the late ORFs of HPV16, was capable of producing infectious virus in organotypic raft cultures. We were interested in determining whether the ability of this chimeric genome to produce infectious virus was the result of HPV18 and HPV16 being similarly oncogenic, anogenital types and whether more disparate PV types could also interact functionally. To test this we created a series of HPV18 chimeric genomes where the ORFs for the HPV18 capsid genes were replaced with the capsid genes of HPV45, HPV39, HPV33, HPV31, HPV11, HPV6b, HPV1a, CRPV, and BPV1. All chimeras were able to produce infectious chimeric viral particles, although with lower infectivity than wild-type HPV18. Steps in the viral life cycle and characteristics of the viral particles were examined to identify potential causes for the decrease in infectivity.

Identification and characterization of the Orf49 protein of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Kaposi's sarcoma is the most common neoplasm among human immunodeficiency virus-positive individuals. Like other herpesviruses, KSHV is able to establish a predominantly latent, life-long infection in its host. The KSHV lytic cycle can be triggered by a number of stimuli that induce the expression of the key lytic switch protein, the replication and transcription activator (RTA) encoded by Orf50. The expression of Rta is necessary and sufficient to trigger the full lytic program resulting in the ordered expression of viral proteins, release of viral progeny, and host cell death. We have characterized an unknown open reading frame, Orf49, which lies adjacent and in the opposite orientation to Orf50. Orf49 is expressed during the KSHV lytic cycle and shows early transcription kinetics. We have mapped the 5' and 3' ends of the unspliced Orf49 transcript, which encodes a 30-kDa protein that is localized to both the nucleus and the cytoplasm. Interestingly, we found that Orf49 was able to cooperate with Rta to activate several KSHV lytic promoters containing AP-1 sites. The Orf49-encoded protein was also able to induce transcriptional activation through c-Jun but not the ATF1, ATF2, or CREB transcription factor. We found that Orf49 could induce phosphorylation and activation of the transcription factor c-Jun, the Jun N-terminal kinase (JNK), and p38. Our data suggest that Orf49 functions to activate the JNK and p38 pathways during the KSHV lytic cycle.

Characterization of Kaposi's sarcoma-associated herpesvirus (KSHV) K1 promoter activation by Rta.

The K1 gene of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 46-kDa transmembrane glycoprotein that possesses transforming properties, initiates signaling pathways in B cells, and prevents apoptosis. Here, we demonstrate a mechanism for activation of the K1 promoter by the Rta transactivator. Electrophoretic mobility shift assay (EMSA) analysis of the K1 promoter demonstrated that purified Rta protein bound to the K1 promoter at three locations, independent of other DNA-binding factors. Transcriptional assays revealed that only two of these Rta DNA-binding sites are functionally significant, and that they could impart Rta responsiveness to a heterologous E4 TATA box promoter. In addition, TATA-binding protein (TBP) bound to a TATA box element located 25 bp upstream of the K1 transcription start site and was also shown to associate with Rta by coimmunoprecipitation analysis. Rta transactivation may therefore be mediated in part through recruitment of TBP to target promoters.

Transcriptional regulation of the K1 gene product of Kaposi's sarcoma-associated herpesvirus.

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) has been shown to be a transforming protein capable of inducing morphological changes and focus formation in rodent fibroblasts. K1 can activate B-cell receptor (BCR) signaling and upregulate activity of the NFAT and NF-kappaB transcription factors. In order to understand the regulation of K1 gene expression, we have analyzed sequences upstream of the K1 gene to identify the K1 promoter element. We have performed 5' rapid amplification of cDNA ends as well as a nuclease protection assay to map the transcriptional start site of the KSHV K1 transcript. The K1 transcriptional start site lies 75 bp upstream of the translation start site. Sequences upstream of the K1 gene were characterized for their ability to activate a luciferase reporter gene in 293 epithelial cells, KSHV-negative B cells (BJAB), KSHV-positive B cells (BCBL-1), and KS tumor-derived endothelial cells (SLK-KS(-)). We found that a 125-bp sequence upstream of the K1 transcript start site was sufficient to fully activate the luciferase reporter gene in all cell types tested. In addition, the viral transcription factor KSHV Orf50/Rta was capable of further activating this promoter element in 293, BJAB, and BCBL-1 cells but not in SLK-KS(-) cells. Promoter constructs containing additional sequences upstream of the 125-bp element did not show further augmentation of transcription in the presence or absence of KSHV Orf50.

Comparison of the Rta/Orf50 transactivator proteins of gamma-2-herpesviruses.

The viral immediate-early transactivator Rta/Orf50 is necessary and sufficient to initiate Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) reactivation from latently infected cells. Since Rta/Orf50 is conserved among all known gamma-2-herpesviruses, we investigated whether the murine gamma-68-herpesvirus (MHV-68) and rhesus monkey rhadinovirus (RRV) homologs can functionally substitute for KSHV Rta/Orf50. (i) Our comparison of 12 KSHV promoters showed that most responded to all three Rta/Orf50proteins, but three promoters (vGPCR, K8, and gB) responded only to the KSHV Rta/Orf50 transactivator. Overall, the activation of KSHV promoters was higher with KSHV Rta than with the RRV and MHV-68 Rta. (ii) Only the primate Rta/Orf50 homologs were able to interfere with human p53-depedent transcriptional activation. (iii) Transcriptional profiling showed that the KSHV Rta/Orf50 was more efficient than it's homologs in inducing KSHV lytic transcription from the latent state. These results suggest that the core functionality of Rta/Orf50 is conserved and independent of its host, but the human protein has evolved additional, human-specific capabilities.