Functional Interactions between Retinoblastoma and c-MYC in a Mouse Model of Hepatocellular Carcinoma

Inactivation of the RB tumor suppressor and activation of the MYC family of oncogenes are frequent events in a large spectrum of human cancers. Loss of RB function and MYC activation are thought to control both overlapping and distinct cellular processes during cell cycle progression. However, how these two major cancer genes functionally interact during tumorigenesis is still unclear. Here, we sought to test whether loss of RB function would affect cancer development in a mouse model of c-MYC-induced hepatocellular carcinoma (HCC), a deadly cancer type in which RB is frequently inactivated and c-MYC often activated. We found that RB inactivation has minimal effects on the cell cycle, cell death, and differentiation features of liver tumors driven by increased levels of c-MYC. However, combined loss of RB and activation of c-MYC led to an increase in polyploidy in mature hepatocytes before the development of tumors. There was a trend for decreased survival in double mutant animals compared to mice developing c-MYC-induced tumors. Thus, loss of RB function does not provide a proliferative advantage to c-MYC-expressing HCC cells but the RB and c-MYC pathways may cooperate to control the polyploidy of mature hepatocytes

Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection

Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria in in vivo mammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation of Rickettsia conorii Malish 7 with the plasmid pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformed R. conorii. Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate that R. conorii(pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformed R. conorii was readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect either in vitro proliferation or in vivo infectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae