Protein Expression Analysis of Chromosome 12 Candidate Genes in Chronic Lymphocytic Leukemia (B-CLL).
Authors: Winkler Dirk , Schneider Christof , Pasqualucci Laura , Brückle Elsbeth , Lichter Peter , Döhner Hartmut , Stilgenbauer Stephan 
Published: April 2020

Abstract
The pathogenic role of trisomy 12 in CLL is unresolved but recent studies indicated an upregulation of chromosome 12 candidate genes on the RNA level. Aim of the current study was to characterize protein expression patterns of chromosome 12 candidate genes when comparing CLL cases with (n=58) or without (n=16) trisomy 12 and three lymphoma cell lines (JVM-2, EHEB, JURKAT). Median clonal tumor load (as defined by FISH or CD5+/CD19+-positivity) was 81%. Western blotting was performed to quantify the protein expression of CKD4, CDK2, Cyclin-D2, p27, Smac, MDM-2, STAT6, ARF3, GLI, Apaf-1 and AID (antibody provided by Cell Signaling Technology, Inc.), all with a gene locus on chromosome 12. An unmutated VH status was observed in 27 of 38 cases in the trisomy 12 group and in 11 of 16 cases in the group with a normal karyotype.
Specific patterns of the investigated proteins could be identified. The most striking differences among the cell lines were found for p27, Cyclin-D2, STAT6, MDM-2 and Smac. Expression of p27 was higher in JURKAT and the CLL samples when compared to EHEB and JVM-2. In contrast, Cyclin-D2 was not expressed by JURKAT and the CLL samples but clearly detectable in EHEB and JVM-2, possibly influenced by EBV-transformation of EHEB and JVM-2. Expression of STAT6 was low in JURKAT when compared to EHEB, JVM-2 and the CLL samples. MDM-2 detection revealed a lower expression of the p57 band in JURKAT as compared to JVM-2 and EHEB which showed all three MDM-2 bands (p90, p76, p57). EHEB had the highest expression of the p57 band of the three cell lines. In CLL patient samples the predominantly detectable MDM-2 bands were p90 and p57 without major differences in expression levels between the investigated subgroups. JURKAT and JVM-2 showed a double band for Smac possibly representing different splice variants. Expression of AID, Smac, ARF-3, Apaf-1 and GLI was as strong in the patient samples as in the cell lines and no difference in expression was seen neither among the CLL cases nor among the cell lines. AID protein expression was very low but detectable in the cell lines as well as in the CLL subgroups without remarkable differences in expression. Therefore, AID protein expression does not seem to be a helpful surrogate marker for VH status or prognosis prediction of CLL patients. CDK4, CDK2 and Cyclin-D2 were generally expressed at lower levels in CLL irrespective of the trisomy 12 status when compared to the cell lines. An earlier study reported an association between high levels of p27 and inferior outcome. The current study confirms a strong expression of p27 in CLL, but all genetic subgroups investigated showed similar levels of p27. Interestingly, recent microarray and RQ-PCR data revealed an up-regulation of the RNA levels of genes located on chromosome 12 (such as p27 and CDK4) in CLL cases with trisomy 12, indicating a gene dosage effect as potential pathomechanism. However, this upregulation of chromosome 12 genes was not observed in the current study on the protein level despite a high tumor load of the samples. This may be due to posttranscriptional mechanisms or a lower sensitivity of Western blotting to detect subtle expression differences. Taken together, none of the candidate gene products investigated appeared to be consistently deregulated as a result of trisomy 12 or VH mutation status in CLL.