bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia.
The bcl-2 gene becomes transcriptionally deregulated in the majority of low-grade non-Hodgkin lymphomas as a result of t(14;18) translocations that place the bcl-2 gene at 18q21 into juxtaposition with the Ig heavy-chain locus at 14q32. This chromosomal translocation or similar bcl-2 gene rearrangements involving the Ig light-chain genes have been reported to occur in some cases of B-cell chronic lymphocytic leukemia (B-CLL). We analyzed the structure, methylation, and expression of the bcl-2 gene in 20 cases of B-CLL or closely related variants of this lymphoproliferative disorder, including at least 16 typical examples of CD5+ B-CLL. None of the 20 specimens had evidence of bcl-2 gene rearrangements, based on Southern blot analysis using three different bcl-2 probes. However, immunoblot analysis using antibodies specific for the Bcl-2 protein showed that 14 of 20 cases (70%) contained levels of p26-Bcl-2 that were equal to or greater than those found in a t(14;18)-bearing lymphoma cell line. Furthermore, in 19 of 20 cases (95%), the Bcl-2 protein was present at levels that were 1.7- to 25-fold higher than in normal peripheral blood lymphocytes. These differences in the relative levels of Bcl-2 protein among cases of B-CLL appeared to be functionally significant, in that a preliminary analysis of 3 representative cases showed that CLL cells with higher levels of Bcl-2 protein survived longer in culture and were delayed in their onset of DNA degradation relative to CLL cells with lower Bcl-2 protein levels. Evaluation of the methylation status of the bcl-2 gene using the isoschizomers Msp I and Hpa II, and a probe corresponding to the first major exon of the gene showed complete demethylation of both copies of the bcl-2 gene in a region corresponding to a 2.4-kb Msp I fragment in all 20 cases of B-CLL. In contrast, analysis of 6 of 6 B-cell lines that harbor a t(14;18) was consistent with hypomethylation of only one of the two bcl-2 alleles. Neither copy of the bcl-2 gene was demethylated in this region in 5 of 5 lymphoid cell lines that lack this translocation. However, hypomethylation of the bcl-2 gene did not necessarily correlate with the relative levels of Bcl-2 protein present in the B-CLL cells, suggesting that additional mechanisms for regulating bcl-2 expression are involved.(ABSTRACT TRUNCATED AT 400 WORDS)
Hanada
M
M
Cancer Research Center, La Jolla Cancer Research Foundation, CA 92037.
Delia
D
D
Aiello
A
A
Stadtmauer
E
E
Reed
J C
JC
D014408:Biomarkers, Tumor / Q000032:analysis*; D002874:Chromosome Mapping; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D006801:Humans; D015151:Immunoblotting; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics*; D008745:Methylation; D009687:Nuclear Proteins / Q000032:analysis; D018809:Proliferating Cell Nuclear Antigen; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D011519:Proto-Oncogenes; D015183:Restriction Mapping; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011505:Protein-Tyrosine Kinases