The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.The life cycle of human papillomaviruses (HPV) is intimately connected to the differentiation program of host epithelial tissues (14,20,22,36). The use of an organotypic (raft) epithelial culture system has allowed for the development of an in vitro culture system capable of reproducing the complete HPV life cycle, including the propagation of infectious viral particles (20,22). The raft culture system has been used to describe in detail the steps in the HPV life cycle (2,8,11,18,(25)(26)(27)(28)(29)(30), including the kinetics and spatial patterns of HPV gene expression (18,(25)(26)(27)(28)(29). Flores et al. used the raft culture system to begin a genetic analysis of the HPV life cycle by using an E7-deficient HPV type 16 (HPV16) genome (7). They found that this genome, while being maintained episomally, failed to amplify its DNA and expressed reduced levels of the L1 capsid protein. That study was done by using a spontaneously immortalized keratinocyte cell line (1). Attempts have been made to use a genetic approach to study the HPV life cycle by using primary keratinocytes (15, 39). These studies found that the majority of mutations examined, both in noncoding and in coding regions, were unstable in their ability to maintain the viral DNA (vDNA) in an episomal state.It has been reported that the interaction of the HPV nonstructural proteins, in particular, E2, with the structural capsid proteins, L1 and L...

This is the first report of a comprehensive multiplex analysis to identify novel VEGF-independent cytokines associated with PDR. Of the 39 inflammatory cytokines tested, 16 are predictive of disease risk, independent of VEGF levels. These PDR-associated cytokines represent potential targets in the treatment of PDR, both in conjunction with anti-VEGF therapy, as well as for patients that are nonresponders to such therapy.

The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.

Tumor cell lines are relied on extensively for cancer investigations, yet cultured cells in an in vitro environment differ considerably in behavior compared with those of the same cancer cells that proliferate and form tumors in vivo. To uncover gene expression changes related to tumor formation, gene expression profiles of human lung adenocarcinoma (A549) cells grown as lung tumors in immune-compromised mice were compared with profiles of the same cells grown in vitro. Additionally, profiles of uninvolved adjacent mouse tissue were determined. A profound interplay between cancer cells and the host was shown that affected a complex protein interaction network involving processes of extracellular interaction, growth factor signaling, hemostasis, immune response, and transcriptional regulation. Growth in vivo of A549 cells, which carry an activating k-ras mutation, induced changes in gene expression that corresponded highly to a pattern characteristic of human lung tumors with k-ras mutation. Cytokines interleukin-4, interleukin-6, and IFN-; each induced distinct in vitro genomic responses in cancer cells that emulated many of the changes in gene expression observed in vivo. Genes that were both selectively induced in vivo and overexpressed in human lung adenocarcinoma tumors included CSPG2, which has not been associated previously with tumor formation. Knockdown in A549 of CSPG2 by RNA interference significantly inhibited tumor growth in vivo but not in vitro. Thus, analysis of tumor xenografts by gene expression profiling has the potential for identifying genes involved in tumor development that may not be expressed in cancer cells grown in vitro.