Effect of pentoxifylline on tumor suppressor and proto-oncogene apoptosis in sperm.
OBJECTIVE
Pentoxifylline (PTX), a methylxanthine phosphodiesterase inhibitor reduces superoxide anions responsible for DNA apoptosis. The null hypothesis was that PTX was equally effective in reducing damage to specific cell genes. The objective was to determine the DNA integrity of the BRCA1 tumor suppressor gene and the c-myc proto-oncogene after PTX.


METHODS
Sperm (64 samples, 4 patients) were preincubated in either 0 (control) or 3.6 mM PTX (30 min), washed and incubated for 4 h at either 37 or 40 degrees C heat shock activation. Single primer polymerase chain reactions (PCR) were carried out on lysed sperm targeting either BRCA1 exon 11 or c-myc exon 1. Control single-stranded DNA (ssDNA) were stained with 9 microM Hoechst 33342 (blue) while PTX-treated ssDNA were stained with SYBR Gold (green). Nytran membrane discs with control ssDNA were hybridized to PTX-derived ssDNA. Fluorescent images stored in a microarray design were analyzed using ANOVA and Students' t-test for (P < 0.05) significance.


RESULTS
BRCA1 integrity was higher with PTX pretreatment (93.3 + 10.4 vs. control 50.5 + 9.2; mean + SEM). In contrast, there was no difference in c-myc integrity (56.8 + 9.0 vs. 41.7 + 6.4). Sense or antisense primers gave similar DNA fragmentation results.


CONCLUSIONS
The data showed PTX pretreatment protected BRCA1 but not c-myc suggesting that PTX did not equally protect different cell genes. A possible explanation was that proto-oncogenes had more fragile sites. The study involved the DNA disc chip assay to assess separate PCR-amplified sense and antisense strands. The results suggested that both strands were equally affected by PTX pretreatment.
Maxwell
David T
DT
Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, California 92354, USA.
Jacobson
John D
JD
King
Alan
A
Chan
Philip J
PJ
D017209:Apoptosis / Q000187:drug effects*; D019313:BRCA1 Protein / Q000187:drug effects; D053938:DNA Fragmentation; D019398:Genes, BRCA1; D016147:Genes, Tumor Suppressor / Q000187:drug effects*; D006801:Humans; D008297:Male; D010431:Pentoxifylline / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects*
D019313:BRCA1 Protein; C000715470:MAS1 protein, human; D010726:Phosphodiesterase Inhibitors; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D010431:Pentoxifylline