Alberto Haces

Genistein-selenium combination induces growth arrest in prostate cancer cells.

The prognosis for patients with metastasized prostate cancer is still poor, despite conventional aggressive therapeutic modalities. Several in vitro studies together with animal models and epidemiological studies have indicated that phytochemicals can be antitumorigenic and may be protective against human cancers. However, the potential antitumor effects of genistein isoflavone, a widely studied nutrient phytochemical, have been equivocal. In this study, we investigated the effects of genistein-selenium (Gn-Se) combination on chemosensitivity and matrix metalloproteinase-2 (MMP-2) expression levels in PC3 (hormone-independent) and LNCaP (hormone-dependent) prostate cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium staining and ATP bioassay showed that genistein, selenium, and Gn-Se combination significantly inhibited growth of LNCaP and PC3 cells in a dose- and time-dependent manner, independent of hormonal status, and with no significant differences in chemosensitivity between LNCaP and PC3. Gn-Se combination induced significantly the greatest growth inhibition in both cell lines. Growth inhibition was through apoptosis induction. The treatment-induced apoptotic cascades are caspase-dependent, with evidence of an alternative non-caspase pathway(s). Treatment also induced a dose- and time-dependent decrease in MMP-2 expression levels in PC3 and LNCaP with no significant differences between the two cells. Gn-Se combination induced the greatest depression in MMP-2. Overall, none of the treatment modalities had any significant inhibitory effect in normal prostate epithelial cells. The data obtained from the present study indicate that Gn-Se combination may have chemopreventive value and/or may be adjuvant to standard therapy for prostate tumors independent of hormonal status. MMP-2 expression in cancer cells has been associated with active invasion and metastasis.

Detection of DNA targets with biotinylated and fluoresceinated RNA probes: Effects of the extent of derivitization on detection sensitivity

The substituted nucleotide aminohexyl-ATP (AH-ATP) was used for synthesis of RNA probes from a plasmid template using the T7 phage promoter. Following synthesis, RNA probes were modified by reaction with N-hydroxysuccinimide (NHS) esters of biotin or fluorescein. Nearest-neighbor analysis was used to quantitate both the incorporation of the substituted nucleotide into RNA and the subsequent modification of the incorporated nucleotide by the NHS esters. The results indicate that AH-ATP is efficiently incorporated into RNA and that modification of the amine group is also efficient. The T7 polymerase shows a bias for ATP over AH-ATP and truncated transcripts are produced if 100% AH-ATP is used for synthesis. However, the use of 50% AH-ATP in the synthesis reaction yields full-length RNA probes that contain on average one amine-labeled nucleotide every 12 bases. This RNA is readily modified by the respective NHS esters to obtain one biotin group per 15-18 total RNA bases or one fluorescein group per 25-35 bases. Probes modified with biotin or fluorescein were used to detect picogram levels of target DNA in a dot blot hybridization format.

DNA sequencing by single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.