BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous collection of leukemic disorders ranging from chemotherapy-sensitive subsets [inversion 16 and t(8;21)], which often can be cured with cytosine arabinoside alone, to the most resistant subsets, which can survive even supralethal levels of combination alkylator chemotherapy (cytogenetic subsets monosomy 5 and monosomy 7). MATERIALS AND METHODS: To analyze the expression of BCL-2 family genes, which are expressed in these subsets of AML, we used PCR sequence amplification reactions that are dependent on oligonucleotide primers representing the BH1 and BH2 homology domains to generate the unique regions between BH1 and BH2. These primers are conserved among all members of the BCL-2 gene family and are separated by a 150 nucleotide region sequence between the BH1 and BH2 domains. The PCR products unique to each BCL-2 family member were cloned directionally into sequencing vectors. The identity of the insert of each clone was determined by slotblots of the DNA amplified from individual colonies and by hybridization with radioactive probes specific to the bcl-2, bcl-x, or bax genes. RESULTS: We found that bcl-2 is the predominant member expressed in AML samples with a poor prognosis (-5, -7), whereas the transcripts of bcl-x are higher than those of bcl-2 in the AML samples with a good prognosis [inv16, t(8;21)]. No significant difference in bax expression was detected between AML subsets of good and bad prognosis. The ratio of bcl-xlong, which inhibits apoptosis, to bcl-xshort, which promotes apoptosis, was determined by amplification with a pair of primers specific to bcl-x followed by separation of the PCR product on agarose gels. Bcl-xlong and bcl-xshort appeared as bands of different molecular mass on a molecular weight gel and were visualized by ethidium bromide staining or Southern blot analysis with a bcl-x-specific probe. CONCLUSIONS: We found that the ratio of bcl-x long to bcl-x short was higher in the AML patients with a poor prognosis. These experiments showed that the levels of BCL-2 family members in the leukemia cells of good- and poor-prognosis subsets are different. In addition, novel members of the BCL-2 family were isolated from the cells of AML patients of either prognosis.
BACKGROUND:Acute myeloid leukemia (AML) is a heterogeneous collection of leukemic disorders ranging from chemotherapy-sensitive subsets [inversion 16 and t(8;21)], which often can be cured with cytosine arabinoside alone, to the most resistant subsets, which can survive even supralethal levels of combination alkylator chemotherapy (cytogenetic subsets monosomy 5 and monosomy 7). MATERIALS AND METHODS: To analyze the expression of BCL-2 family genes, which are expressed in these subsets of AML, we used PCR sequence amplification reactions that are dependent on oligonucleotide primers representing the BH1 and BH2 homology domains to generate the unique regions between BH1 and BH2. These primers are conserved among all members of the BCL-2 gene family and are separated by a 150 nucleotide region sequence between the BH1 and BH2 domains. The PCR products unique to each BCL-2 family member were cloned directionally into sequencing vectors. The identity of the insert of each clone was determined by slotblots of the DNA amplified from individual colonies and by hybridization with radioactive probes specific to the bcl-2, bcl-x, or bax genes. RESULTS: We found that bcl-2 is the predominant member expressed in AML samples with a poor prognosis (-5, -7), whereas the transcripts of bcl-x are higher than those of bcl-2 in the AML samples with a good prognosis [inv16, t(8;21)]. No significant difference in bax expression was detected between AML subsets of good and bad prognosis. The ratio of bcl-xlong, which inhibits apoptosis, to bcl-xshort, which promotes apoptosis, was determined by amplification with a pair of primers specific to bcl-x followed by separation of the PCR product on agarose gels. Bcl-xlong and bcl-xshort appeared as bands of different molecular mass on a molecular weight gel and were visualized by ethidium bromide staining or Southern blot analysis with a bcl-x-specific probe. CONCLUSIONS: We found that the ratio of bcl-x long to bcl-x short was higher in the AMLpatients with a poor prognosis. These experiments showed that the levels of BCL-2 family members in the leukemia cells of good- and poor-prognosis subsets are different. In addition, novel members of the BCL-2 family were isolated from the cells of AMLpatients of either prognosis.
Authors: S N Farrow; J H White; I Martinou; T Raven; K T Pun; C J Grinham; J C Martinou; R Brown Journal: Nature Date: 1995-04-20 Impact factor: 49.962
Authors: Jacqueline C Shultz; Ngoc Vu; Michael D Shultz; Mba-Uzoma U Mba; Brian A Shapiro; Charles E Chalfant Journal: Mol Cancer Res Date: 2012-04-20 Impact factor: 5.852
Authors: Brian A Shapiro; Ngoc T Vu; Michael D Shultz; Jacqueline C Shultz; Jennifer A Mietla; Mazen M Gouda; Adly Yacoub; Paul Dent; Paul B Fisher; Margaret A Park; Charles E Chalfant Journal: J Biol Chem Date: 2016-08-12 Impact factor: 5.157