Human retinoblastoma is not caused by known pRb-inactivating human DNA tumor viruses.

Retinoblastomas occur as the consequence of inactivation of the tumor suppressor retinoblastoma protein (pRb), classically upon biallelic inactivation of the RB1 gene locus. Recently, human papillomavirus (HPV) genomic DNA has been detected in retinoblastomas. To investigate the possibility that oncoproteins encoded by pRb-inactivating DNA tumor viruses play a role in the pathogenesis of human retinoblastoma, 40 fresh-frozen tumors were analyzed for the presence of HPV, adenovirus (HAdV) and polyomavirus (BKV, JCV and SV40) genomic DNA sequences by real-time polymerase chain reaction (PCR). Tumors were screened for genetic and epigenetic alterations in all 27 exons of the RB1 gene locus and promoter by exonic copy number detection, sequencing and methylation-specific PCR of the promoter region. Retinoblastoma tumors from children with bilateral familial (n=1), bilateral nonfamilial (n=1) and unilateral nonfamilial (n=38) disease were analyzed. Inactivating modifications to the RB1 gene locus were identified on both the alleles in 27 tumors, one allele in 8, and neither allele in 5 cases. A median of over 107,000 tumor cells were analyzed for viral genomic DNA in each PCR reaction. All tumor samples were negative for 37 HPV types, 51 HAdV types, BKV and JCV genomic sequences. Very low copy number (0.2-260 copies per 100,000 tumor cells) SV40 genomic DNA detected in 8 of 39 samples was demonstrated to be consistent with an artifact of plasmid-derived SV40. In contrast to recent reports, we obtained substantial quantitative evidence indicating that neither HPV nor any other pRb-inactivating human DNA tumor viruses play a role in the development of retinoblastoma, regardless of RB1 genotype.

Relationship between prevalent oral and cervical human papillomavirus infections in human immunodeficiency virus-positive and -negative women.

Human papillomavirus (HPV) is an etiologic agent for both oropharyngeal and cervical cancers, yet little is known about the interrelationship between oral and cervical HPV infections. Therefore, we compared the prevalences and type distributions of oral and cervical HPV infections and evaluated infection concordance in a cross-sectional study within the Women's Interagency HIV Study cohort. Oral rinse and cervical-vaginal lavage samples were concurrently collected from a convenience sample of 172 human immunodeficiency virus (HIV)-positive and 86 HIV-negative women. HPV genomic DNA was detected by PGMY09/11 L1 consensus primer PCR and type specified by reverse line blot hybridization for 37 HPV types and beta-globin. Only 26 of the 35 HPV types found to infect the cervix were also found within the oral cavity, and the type distribution for oral HPV infections appeared distinct from that for cervical infections (P<0.001). Oral HPV infections were less common than cervical infections for both HIV-positive (25.2% versus 76.9%, P<0.001) and HIV-negative (9.0% versus 44.9%, P<0.001) women. Oral HPV infections were more common among women with a cervical HPV infection than those without a cervical HPV infection (25.5% versus 7.9%, P=0.002). The majority of women (207; 93.7%) did not have simultaneous oral and cervical infections by the same HPV type; however, the number of women who did (14; 6.3%) was significantly greater than would be expected by chance (P=0.0002). Therefore, the oral and cervical reservoirs for HPV infection are likely not entirely independent of one another.

Sim2 contributes to neuroendocrine hormone gene expression in the anterior hypothalamus.

Paraventricular (PVN) and supraoptic nuclei of the hypothalamus maintain homeostasis by modulating pituitary hormonal output. PVN and supraoptic nuclei contain five major cell types: oxytocin-, vasopressin-, CRH-, somatostatin-, and TRH-secreting neurons. Sim1, Arnt2, and Otp genes are essential for terminal differentiation of these neurons. One of their common downstream genes, Brn2, is necessary for oxytocin, vasopressin, and CRH cell differentiation. Here we show that Sim2, a paralog of Sim1, contributes to the expression of Trh and Ss genes in the dorsal preoptic area, anterior-periventricular nucleus, and PVN. Sim2 expression overlaps with Trh- and Ss-expressing cells, and Sim2 mutants contain reduced numbers of Trh and Ss cells. Genetically, Sim1 acts upstream of Sim2 and partially compensates for the loss of Sim2. Comparative expression studies at the anterior hypothalamus at early stages reveal that there are separate pools of Trh cells with distinctive molecular codes defined by Sim1 and Sim2 expression. Together with previous reports, our results demonstrate that Sim1 and Otp utilize two common downstream genes, Brn2 and Sim2, to mediate distinctive sets of neuroendocrine hormone gene expression.

Identification of the downstream targets of SIM1 and ARNT2, a pair of transcription factors essential for neuroendocrine cell differentiation.

SIM1 and ARNT2 are two basic helix-loop-helix/PAS (Per-Arnt-Sim) transcription factors that control the differentiation of neuroendocrine lineages in the mouse hypothalamus. Heterozygous Sim1 mice also develop early onset obesity, possibly due to hypodevelopment of the hypothalamus. Although SIM1 and ARNT2 form heterodimers to direct the same molecular pathway, knowledge of this pathway is limited. To facilitate the identification of their downstream genes, we combined an inducible gene expression system in a neuronal cell line with microarray analysis to screen for their transcriptional targets. This method identified 268 potential target genes of SIM1/ARNT2 that displayed >1.7-fold induced expression. 15 of these genes were subjected to Northern analysis, and a high percentage of them were confirmed to be up-regulated. In vivo, several of these genes showed neuroendocrine hypothalamic expression correlating with that of Sim1. Furthermore, we found that expression of two of these potential targets, the Jak2 and thyroid hormone receptor beta2 genes, was lost in the neuroendocrine hypothalamus of the Sim1 mutant. The expression and predicted functions of many of these genes provide new insight into both the Sim1/Arnt2 action in neuroendocrine hypothalamus development and the molecular basis for the Sim1 haplo-insufficient obesity phenotype.

Sim2 mutants have developmental defects not overlapping with those of Sim1 mutants.

The mouse genome contains two Sim genes, Sim1 and Sim2. They are presumed to be important for central nervous system (CNS) development because they are homologous to the Drosophila single-minded (sim) gene, mutations in which cause a complete loss of CNS midline cells. In the mammalian CNS, Sim2 and Sim1 are coexpressed in the paraventricular nucleus (PVN). While Sim1 is essential for the development of the PVN (J. L. Michaud, T. Rosenquist, N. R. May, and C.-M. Fan, Genes Dev. 12:3264-3275, 1998), we report here that Sim2 mutant has a normal PVN. Analyses of the Sim1 and Sim2 compound mutants did not reveal obvious genetic interaction between them in PVN histogenesis. However, Sim2 mutant mice die within 3 days of birth due to lung atelectasis and breathing failure. We attribute the diminished efficacy of lung inflation to the compromised structural components surrounding the pleural cavity, which include rib protrusions, abnormal intercostal muscle attachments, diaphragm hypoplasia, and pleural mesothelium tearing. Although each of these structures is minimally affected, we propose that their combined effects lead to the mechanical failure of lung inflation and death. Sim2 mutants also develop congenital scoliosis, reflected by the unequal sizes of the left and right vertebrae and ribs. The temporal and spatial expression patterns of Sim2 in these skeletal elements suggest that Sim2 regulates their growth and/or integrity.