No abstract

Cell cycle kinase inhibitors have advanced into clinical trials in oncology. One such molecule, JNJ-7706621, is a broad-spectrum inhibitor of the cyclin-dependent kinases and Aurora kinases that mediate G 2 -M arrest and inhibits tumor growth in xenograft models. To determine the putative mechanisms of resistance to JNJ-7706621 that might be encountered in the clinic, the human epithelial cervical carcinoma cell line (HeLa) was exposed to incrementally increasing concentrations of JNJ-7706621. The resulting resistant cell population, designated HeLa-6621, was 16-fold resistant to JNJ-7706621, cross-resistant to mitoxantrone (15-fold) and topotecan (6-fold), and exhibited reduced intracellular drug accumulation of JNJ-7706621. ABCG2 was highly overexpressed at both the mRNA (f163-fold) and protein levels. The functional role of ABCG2 in mediating resistance to JNJ-7706621 was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C, restored the sensitivity of HeLa-6621 cells to JNJ-7706621 and to mitoxantrone; (b) human embryonic kidney-293 cells transfected with ABCG2 were resistant to both JNJ-7706621 and mitoxantrone; and (c) resistant cells that were removed from the drug for 12 weeks and reverted to susceptibility to JNJ-7706621 showed near-normal ABCG2 RNA levels. ABCG2 is likely to limit the bioavailability of JNJ-7706621 because oral administration of JNJ-7706621 to Bcrp (the murine homologue of ABCG2) knockout mice resulted in an increase in the plasma concentration of JNJ-7706621 compared with wild-type mice. These findings indicate that ABCG2 mediates the resistance to JNJ-7706621 and alters the absorption of the compound following administration.

The retroviral integrase (IN) carries out the integration of viral DNA into the host genome. The IN protein consists of three domains: the N-terminal HHCC motif, the catalytic core region, and the C-terminus. The Moloney murine leukemia virus (M-MuLV) IN encodes a unique 45-amino-acid domain N-terminal to the HHCC motif. The function of the N-terminus of M-MuLV IN was studied through deletional and mutational analyses. The IN 1-105 domain was dissected into two halves expressing either the unique N-terminus or the HHCC domain. Although the parental IN 1-105 could functionally complement the core-C-terminus for integration reactions, neither half of the N-terminus was sufficient. Partial complementation of strand transfer, but not 3prime prime or minute processing, could be obtained through mixing the two halves. The dimerization of the M-MuLV N-terminus was dependent on the expression of the intact 1-105. Critical basic amino acids within the HHCC domain which are required for 3' processing and strand transfer reactions were identified through alanine mutagenesis. Loss of in vitro strand transfer activity correlated with loss of viral titer in vivo for this cluster of basic amino acids within the HHCC domain.

Moloney murine leukemia virus (M-MuLV) proviruses carrying integrase (IN) protein tagged either with a simian virus 40 (SV40) nuclear localization signal (NLS) or various antigenic epitopes were generated. Hexahistidine (His(6)), hemagluttinin (HA), or two consecutive HA sequences (2XHA) were fused to the C-terminus of IN as antigenic markers. These epitope-tagged IN proteins were stably expressed through multiple rounds of infection. The IN-His(6), IN-HA, and IN-2XHA proteins, purified from virus, could be immunoprecipitated with antibodies against His(6) and HA, respectively. An M-MuLV provirus encoding the SV40 large T antigen NLS fused to IN at the same position as the epitope tags was also passaged through cells. In contrast to the stability of the epitope tags, the SV40 NLS sequence was rapidly mutated by a frameshift mutation that introduced negatively charged amino acids into the basic NLS. The instability of the NLS suggests that the strong nuclear localization of the IN-SV40 NLS may have detrimental effects on virus assembly. These observations have implications for studying nuclear transport properties of M-MuLV and for engineering a murine-based retroviral vector for gene therapy.

Following entry into the cytoplasm of the host cell, reverse transcription of the retroviral RNA genome into doublestranded viral DNA occurs within large nucleoprotein complexes derived from the core of the infecting virion, termed preintegration complexes (PICs). These complexes must then gain access to host cell chromosomes within the nucleus to achieve stable integration. Human immunodeficiency virus type 1 (HIV-1) can infect certain types of nondividing cells (5,22,46,74). Although the ability of lentiviruses to infect nondividing cells has been exploited in the development of vectors for human gene therapy, their application is limited by safety concerns. In contrast, nuclear accumulation of Moloney murine leukemia virus (Mo-MuLV) viral DNA increases only after infected cells pass through mitosis (61). This property limits the range of cells and target tissues that can use murinebased retroviral vectors for gene transfer or gene therapy.Many studies put forth the model that HIV-1 PICs are actively imported into the nucleus. In support of this model, nuclear localization signals (NLSs) have been reported on a number of viral proteins that comprise the HIV-1 PIC. The role or roles that these sequences play in viral replication, however, remain unclear. In addition, a cis-acting viral DNA structure generated during lentivirus-specific reverse transcription, termed the central DNA flap, may also be involved in the nuclear import of the HIV-1 genome (77). To date, no NLSs have been identified in MuLV PIC components. This suggests that it is the disassembly of the nuclear envelope during mitosis that renders the chromosomes accessible to the MuLV PIC.Interestingly, the avian sarcoma virus (ASV), which has a novel NLS sequence (44, 45), presents an intermediate phenotype in which mitosis-independent replication can occur, but at a lower efficiency than HIV-1 (31).Previous analysis indicated various molecular tags placed at the C terminus of the Mo-MuLV integrase (IN) protein could be incorporated into viral particles when expressed within the context of a replication-competent virus. IN proteins tagged with either six-His or hemagglutinin (HA) epitope tags were shown to be genetically stable during replication. In contrast, insertion of the SV40 large T-antigen NLS (SV40 NLS) was not stable, resulting in the selection and propagation of viruses in which the basic NLS sequence was disrupted (65). This study examines the stability of alternative NLS sequences within Mo-MuLV IN and the influence the NLS sequences have on the nuclear transport properties of Mo-MuLV. A similar approach to alter nuclear transport properties was reported to be successful for spleen necrosis virus (SNV), in which the efficiency of infecting nondividing cells was altered by incorporating an HIV-1 matrix (MA) protein NLS sequence into the SNV MA protein (56).Expression of IN and IN-SV40 NLS proteins. The effects of inserting the SV40 NLS into M-MuLV IN were first examined in an IN expression system in the absence of additional viral protein...