Protein expression analysis of chromosome 12 candidate genes in chronic lymphocytic leukemia (CLL)

Abstract Trisomy 12 is the most common cytogenetic abnormality in chronic lymphocytic leukemia (CLL), and a number of studies have suggested that it may be an adverse prognostic indicator. We have evaluated the usefulness of fluorescence in situ hybridization with a chromosome 12- specific probe as a simple means for detecting trisomy 12 in interphase cells. Forty cases of B-cell CLL previously studied with conventional cytogenetic techniques were analyzed with a biotinylated probe to the centromeric region of chromosome 12. Thirty of these retrospective cases could be reevaluated with in situ hybridization. Our analysis showed three hybridization signals (ie, trisomy 12) in interphase cells from seven of seven cases found previously to have trisomy 12. Trisomy 12 was also detected in five additional cases: in one case thought to have a normal karyotype, in two cases that had been inadequate for routine cytogenetic analysis, and in two cases that had been found to have an abnormal karyotype without trisomy 12. In a prospective series of 20 newly accrued CLL cases, all cases were analyzed successfully by in situ hybridization and six (30%) showed trisomy 12. We were able to perform the analysis on routinely prepared and previously Wright- stained peripheral blood smears. We conclude that fluorescence in situ hybridization is a simple means for the detection of trisomy 12 in CLL. The technique is more sensitive than conventional cytogenetic analysis and would be a useful tool in clinical studies.

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Protein Expression Patterns of Candidate Genes Involved in Apoptosis and Cell Cycle Control in Genetic Subgroups of Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.2796.2796 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2796-2796

Author(s):

Christof Schneider ◽

Dirk Winkler ◽

Meike Loddenkemper ◽

Alexander Krober ◽

Peter Lichter ◽

...

Keyword(s):

Cell Cycle ◽

Protein Expression ◽

Cyclin D1 ◽

Candidate Genes ◽

Cell Lines ◽

Normal Karyotype ◽

Lymphocytic Leukemia ◽

Expression Levels ◽

Protein Levels ◽

Atm Protein

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course. Genomic aberrations (such as 13q−, 11q−, +12q, 17p−) can be found in about 80% of CLL cases and define pathogenic as well as clinical subgroups. Similarly, the mutational status of the variable region of the immunoglobulin heavy-chain gene (VH) identifies subgroups with different maturation stage and clinical outcome. In this study protein expression levels of candidate genes involved in cell cycle and apoptosis control (p53, ATM, Akt1, PI3-K, p21, p27, cdk4, Cyclin-D1, D2, D3, Bax, Bcl-2, Apaf-1, Smac, XIAP, cIAP2, survivin) were examined by Western Blotting. A total of 87 CLL cases derived from the subgroups with 11q- (n=22), 17p-/p53 mutation (n=18), +12q (n=24), 13q- (n=8) or a normal karyotype (n=15) were studied and compared to the cell lines EHEB and JVM-2. VH-mutation status was available for 65 cases (unmutated n=48, mutated n=17). Due to limitations in sample availability not all proteins could be examined in all cases. A highly homogenous expression pattern for all the proteins studied was observed in the CLL subgroup with a normal karyotype. This pattern was independent of the VH-status. CLL samples with normal karyotype, +12q and 13q deletion showed equal levels of ATM as compared to EHEB and JVM-2. As compared to cases with a normal karyotype the ATM level within the 11q- subgroup was reduced in 5 cases and absent in 1 case among 11 evaluable 11q- cases. The 17p- subgroup was comprised of 3 cases with concomitant 17p- and 11q- and 15 cases with 17p- but no 11q-. The latter group showed ATM protein levels comparable to the levels of the normal karyotype group. In the group with 17p- and 11q- there was an ATM expression level similar to the groups with 17p- and normal karyotype in two cases while one case had a reduced ATM protein level comparable to the 11q- subgroup. All cases with 17p- exhibited a stronger expression of p53 as compared to the cell lines and all other cases, except for one case with normal karyotype and one with an 11q-. No p53 mutations could be detected in exons 5–9 by sequencing in these two cases. High levels of survivin protein were found in all cases with 17p- and/or 11q-, 13q-, +12q while the subgroup with a normal karyotype showed lower levels. High levels of cdk4 protein were expressed in cases with 17p-, 11q- and 13q- while cdk4 protein levels were low in the subgroup with +12q and normal karyotype. Regarding p21, p27, Bcl2, Bax, Smac, Apaf-1, Cyclin D1–D3, cIAP2, XIAP, Akt1 and PI3K no variation in the expression levels were observed across the genetic CLL subgroups. Comparing the CLL cases to the cell lines the differences in expression levels were found for the cell cycle regulators Cyclin D1, D2, D3, p21 and p27. While the cell lines showed strong protein levels for Cyclin D1, D2, D3 and p21, they were nearly absent in the CLL cases. Expression of p27 was higher in all CLL cases as compared to JVM-2 and EHEB. In conclusion, the 17q- subgroup was the only group with a high level of p53 protein expression indicating that p53 is the affected gene in this subgroup. In contrast, the ATM protein levels are reduced only in a part of the 11q- cases indicating a possible role of additional candidate genes. Cases with +12q and normal karyotype showed weak expression of cdk4 pointing out a possible function in these subgroups.

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Relationship between nitrogen mustard drug resistance in B-cell chronic lymphocytic leukemia (B-CLL) and protein expression of Bcl-2, Bax, Bcl-X and p53

Cancer Letters ◽

10.1016/s0304-3835(97)00336-4 ◽

1997 ◽

Vol 121 (1) ◽

pp. 59-67 ◽

Cited By ~ 12

Author(s):

G Christodoulopoulos ◽

N Fotouhi ◽

S Krajewski ◽

J.C Reed ◽

M Alaoui-Jamali ◽

...

Keyword(s):

Drug Resistance ◽

Chronic Lymphocytic Leukemia ◽

Protein Expression ◽

B Cell ◽

Nitrogen Mustard ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

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Lyn Kinase Contributes to the Reprogramming of Fibroblasts Promoting Chronic Lymphocytic Leukemia Progression

Blood ◽

10.1182/blood-2019-129299 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4283-4283

Author(s):

Alexander Frederik Vom Stein ◽

Maximilian Koch ◽

Sebastian Reinarzt ◽

Anna Lucas ◽

Rocio Rebollido-Rios ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Protein Expression ◽

Cell Survival ◽

Lymphocytic Leukemia ◽

Specific Cell ◽

Deficient Mouse ◽

Lyn Kinase ◽

Drug Concentrations ◽

Gene And Protein Expression ◽

Functional Relevance

Growth of chronic lymphocytic leukemia cells strongly depends on a nurturing microenvironmental niche that is specifically primed by diverse, bi-directional interactions to promote leukemic homing, proliferation and progression. The Src-family kinase Lyn was previously identified by our group as a key factor for the formation of this pro-leukemic niche and for the expansion of CLL cells, using the Eµ-TCL1 mouse model. In order to attribute the pro-leukemic function of Lyn to a specific cell type, chimeric mice with lineage-specific defects of Lyn within hematopoietic or non-hematopoietic compartments were generated by irradiating BL6J-mice lethally and restoring their hematopoietic system with Lyn-WT or Lyn-KO stem cells. Consecutively mice were xenotransplanted with TCL1+-malignant cells. Lyn deficiency within the non-hematopoietic compartment decelerated leukemic expansion to a higher degree than did Lyn deficiency within the hematopoietic compartment. Completely Lyn deficient mice showed a more prominent retardation of leukemic expansion compared with both lineage specific Lyn deficient mouse strains, suggesting an additive effect of the two distinct compartments for leukemic expansion. In focusing on the non-hematopoietic fibroblastic bystander cells, primary human CLL cells were cocultured in vitro with Lyn-deficient mouse embryonic fibroblasts as well as Lyn-KO human HS5 cells, generated via the CRISPR-Cas9 system, and leukemic cell survival was assessed over time. All Lyn-deficient fibroblasts showed a significantly reduced feeding capacity for CLL cells compared to WT stroma, indicating the functional relevance of Lyn in leukemia-associated fibroblasts. Subsequently, transcriptomic, proteomic and phosphoproteomic alterations related to Lyn-KO in HS5 cells were comprehensively analyzed, revealing a surprisingly extensive change in gene and protein expression pattern that appeared to be regulated mainly at the transcriptional level. The differentially expressed genes were remarkably often extracellular matrix (ECM)-, cytoskeleton- or cytokine-associated. GO-term enrichment analysis additionally suggested a correlation with ECM processes. Therefore, we hypothesized that Lyn-deficiency might induce transcriptional changes of the cancer-associated fibroblast (CAF)-like phenotype, thus leading to a reduction in leukemic feeding capacity. The diminished expression of several CAF-makers congruent with this reduced activation status was validated in Lyn-KO fibroblasts, as well as the transcriptionally regulated differential expression of chosen target genes. Amongst those, the deubiquitinating enzyme UCHL1 was most abundantly reduced in Lyn-KO HS5 cells, showing an almost complete loss of mRNA and protein expression. Application of a specific UCHL1-inhibitor -in a dose without toxic effects on CLL cells - to CLL-stroma coculture resulted in a significantly hampered feeder effect and reduced CLL cell survival, implying a functional relevance of microenvironmental UCHL1 for stromal support in our system. Additionally, stroma cell death induced by higher drug concentrations in WT cells was completely prohibited in Lyn-KO stroma, illustrating the importance of Lyn for regulating UCHL1 expression and function. In summary, we propose that the Lyn kinase contributes to the formation of a supportive microenvironment via the transcriptional reprogramming of stroma fibroblasts into a "CAF-like" phenotype, which echances viability of CLL cells. In addition, UCHL1 might be a potentially druggable mediator of this activation process. Disclosures Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.

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