mRNA expression analysis of a variety of apoptosis-related genes, including the novel gene of the BCL2-family, BCL2L12, in HL-60 leukemia cells after treatment with carboplatin and doxorubicin

Abstract

Apoptosis is a type of programmed cell death involved in many crucial biological processes. It represents the basic mechanism for the action of chemotherapeutic agents, such as doxorubicin and carboplatin. Both are able to cause cell death through the induction of apoptosis in the human leukemic cell line HL-60. We investigated the possible alterations in the expression of apoptosis-related genes, including the novel BCL2L12 gene, which was recently cloned in our group. The kinetics of apoptosis induction and cell toxicity was investigated by DNA laddering and by the MTT method, respectively. Total RNA was extracted and cDNA was prepared by reverse transcription. BCL2 , BAX , FAS , caspase-9, caspase-3 and BCL2L12 were amplified by PCR. Overexpression of FAS , BCL2L12 and caspase-3 was observed after treatment of HL-60 cells for 3 or 6 h with carboplatin, while their expression was decreased after a 12-h treatment, demonstrating that these genes may take part in the early stages of apoptosis. Overexpression of the same genes was also observed after 6 h of treatment with doxorubicin (concomitantly with DNA laddering). In the case of carboplatin-induced apoptosis we detected down-regulation of BAX , BCL2 and caspase-9, whereas in the case of doxorubicin, BAX and BCL2 remained at control levels and caspase-9 was increased.

Treatment of Gastric Cancer Cells with 5-Fluorouracil/Leucovorin and Irinotecan Induces Distinct Alterations in the mRNA Expression of the Apoptosis-Related Genes, Including the Novel Gene <i>BCL2L12</i>

“…BCL2 mRNA levels also varied in breast cancer cell lines, mainly in the MCF-7, after treatment with anticancer drugs; predominantly benzo-(a)pyrene, 5-FU, tamoxifen, adriamycin, etoposide, camptothecin, Taxol and cisplatin [24] . Modulations were also observed in HL-60 leukemic cells after treatment with topotecan and methotrexate [25] , as well as carboplatin and doxorubicin [26] .…”

“…These mutations are functional and they inhibit the proapoptotic activity of the protein, thus contributing to carcinogenesis [27] . Gene expression of BAX at the mRNA level has been detected to fluctuate in the breast cancer cell line MCF-7 after being exposed to chemotherapeutic agents, such as benzo-(a)pyrene, lindane, adriamycin, etoposide, camptothecin, Taxol and cisplatin [24] , and additionally in the HL-60 cells after treatment with carboplatin [26] and etoposide [28] .…”

“…BCL2L12 expression levels have been analyzed in breast and colon cancer [35][36][37] and also in HL-60 cells following treatment with chemotherapeutic drugs, such as cisplatin, carboplatin, doxorubicin [26,38] , etoposide [28] , topotecan and methotrexate [25] . Moreover, BCL2L12 expression has been tested in MCF-7 cells after treatment with etoposide, taxol [39] , cisplatin, carboplatin and doxorubicin [40] .…”

“…The novel gene BCL2L12 seemed to follow the expressional pattern of BCL2 , but not in an absolute manner. Though, as both anti-and proapoptotic proteins can be downregulated in drug-induced apoptosis [26] , the down-or upregulation of BCL2L12 in a variety of cell lines and in response to nonsimilar antitumor drugs could not directly indicate its anti-or proapoptotic function.…”

The Novel Member of the BCL2 Gene Family, BCL2L12, Is Substantially Elevated in Chronic Lymphocytic Leukemia Patients, Supporting Its Value As a Significant Biomarker

“…In vitro studies in human leukemia cell lines have revealed notable alterations of BCL2L12 mRNA expression in HL-60 acute promyelocytic leukemia cells after treatment with various chemotherapeutic drugs, including cisplatin, carboplatin, doxorubicin, methotrexate, etoposide, topotecan, vincristine, and taxol [27][28][29][30][31], as well as in SHI-1 acute monocytic leukemia cells after treatment with bortezomib [32]. Recently, it has also been proposed that BCL2L12 and BCL2L12-A may play an important role in cisplatin-induced apoptosis in MDA-MB-231 breast adenocarcinoma cells [33,34].…”