Replication from oriP of Epstein-Barr virus requires human ORC and is inhibited by geminin.

A hypomorphic mutation made in the ORC2 gene of a human cancer cell line through homologous recombination decreased Orc2 protein levels by 90%. The G1 phase of the cell cycle was prolonged, but there was no effect on the utilization of either the c-Myc or beta-globin cellular origins of replication. Cells carrying this mutation failed to support the replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV), and this defect was rescued by reintroduction of Orc2. Orc2 specifically associates with oriP in cells, most likely through its interaction with EBNA1. Geminin, an inhibitor of the mammalian replication initiation complex, inhibits replication from oriP. Therefore, ORC and the human replication initiation apparatus is required for replication from a viral origin of replication.

Amplification of the translocated c-myc genes in three Burkitt lymphoma cell lines.

Translocations of the coding exons of the human c-myc gene are consistent features of human Burkitt lymphomas (BL). In the BL cell lines CA46, JD40, and ST486, the second and third c-myc exons have been translocated into the immunoglobulin heavy chain locus. In addition to this rearrangement, in all three cell lines, we have found that the translocated c-myc exons show low-level amplification relative to restriction fragments from the germ-line c-myc gene. The patterns of hybridization of an IgM switch region probe suggest that immunoglobulin heavy chain sequences have been co-amplified with the translocated c-myc sequences. Differential sedimentation was used to determine whether the amplified sequences reside in high-molecular-weight chromosomes or low-molecular-weight extrachromosomal DNA. In JD40 and ST486 cells, the amplified c-myc sequences were found on high-molecular-weight chromosomes ST486 cells also contained translocated C-myc sequences in low-molecular-weight, extrachromosomal DNA, as did CA46 cells. These conclusions were corroborated by fluorescence in-situ hybridization (FISH) of HeLa, CA46, ST486 and JD40 metaphase chromosomes. These results suggest that there is ongoing selection for cells containing amplified copies of the expressed c-myc sequences. and that there is continuous generation of extrachromosomal copies of the translocated c-myc sequences in ST486 and CA46 cells.

Spermatogenic events in rat and mice following intratesticular administration of optical isomers of gossypol.

Male rats and mice were administered racemic and dextrorotatory gossypol intratesticularly at the dose of 200 micrograms/testis. In a separate experiment, 100 micrograms of dextrorotatory and levorotatory gossypol was administered to male mice. Animals were sacrificed 72 hours after drug treatment. In another experiment, mice were sacrificed 3, 10, 20, 30 and 60 days after racemic gossypol (200 micrograms/testis) administration. Marked degenerative changes in rat and mice tests were observed in the animals receiving racemic gossypol. Dextrorotatory gossypol had less pronounced effect, both in rat and mice. Stage 7 and 16 spermatids were most susceptible to the deleterious effects of racemic gossypol. In mice, 57.52% and 85.40% decrease in stage 7 and 16 spermatids were observed 72 hrs after drug administration. A progressive recovery was observed after drug treatment; the damage (stage 7 & 16) was reduced to 7.92% and 21.30% after 60 days. Histopathological changes in mice testis following 100 micrograms of levorotatory gossypol were distinctly different from those of the dextrorotatory (100 micrograms/testis) gossypol. On the basis of observations made by us, it can be suggested that mice equally respond to the antifertility effect of gossypol following intratesticular administration. The dextrorotatory gossypol, both in rat and mice, had less pronounced effect on the histoarchitecture of the testis in comparison to racemic and levorotatory gossypol. Our observations further suggest that this animal model provides meaningful information on the mechanism of action of gossypol, and recovery of spermatogenesis is possible after termination of drug treatment.