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The D cyclins are important cell cycle regulatory proteins involved in the pathogenesis of some lymphomas. Cyclin D1 overexpression is a hallmark of mantle cell lymphoma, whereas cyclins D2 and D3 have not been shown to be closely associated with any particular subtype of lymphoma. In the present study, we found that cyclin D2 was specifically overexpressed in the proliferation centers (PC) of all cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) examined (19/19). To examine the molecular mechanisms underlying this overexpression, we immunohistochemically examined the expression of nuclear factor (NF)-κB, p15, p16, p18, and p27 in the PC of six patients. Five cases showed upregulation of NF-κB expression, which is known to directly induce cyclin D2 by binding to the promoter region of CCND2. All six PC examined demonstrated downregulation of p27 expression. In contrast, upregulation of p15 expression was detected in five of six PC examined. This discrepancy suggests that unknown cell cycle regulatory mechanisms involving NF-κB-related pathways are also involved, because NF-κB upregulates cyclin D2 not only directly, but also indirectly through c-Myc, which is believed to downregulate both p27 and p15. In conclusion, cyclin D2 is overexpressed in the PC of CLL/SLL and this overexpression is due, in part, to the upregulation of NF-κB-related pathways. (Cancer Sci 2011; 102: 2103–2107)
The D-type cyclins (D1, D2, and D3) play key roles in cell cycle machinery and the biochemical functions of these cyclins partially overlap.(1,2) The D cyclins positively regulate cell proliferation by binding to cyclin-dependent kinase (CDK) 4 and CDK6, resulting in the phosphorylation of the retinoblastoma protein and the G1/S transition of the cell.(2,3)
Dysregulation of D cyclins has been implicated in the pathogenesis of lymphoid malignancies.(4) Among the three D cyclins, the expression pattern of cyclin D1 has been well documented;(5) indeed, overexpression of cyclin D1 induced by translocation of t(11;14)(q13;q32) serves as a hallmark for the diagnosis of mantle cell lymphoma.(6) Recent studies have identified a rare type of cyclin D1-negative mantle cell lymphoma that overexpresses cyclin D2 or D3.(7,8) Cyclins D2 and D3 are also detected in various lymphomas,(9,10) but these two proteins are not as specific to certain lymphomas as is cyclin D1.(10)
Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is an indolent lymphoma/leukemia.(11) At least two major types of CLL/SLL are known to exist, one with mutated immunoglobulin heavy-chain variable region (IGHV) genes, which is negative for zeta-chain-associated protein kinase 70 (ZAP-70), and the other with unmutated IGHV genes, which is positive for ZAP-70.(11–14)
Previous reports have described that peripheral blood neoplastic cells of patients with CLL/SLL are closely related to cyclin D2.(15–17) In these studies, the vast majority of circulating tumor cells were arrested in the G0/early G1 phase and were not the proliferative component.(11) The proliferating cells are located in the proliferation centers (PC) of lymph nodes, which show a pseudofollicular pattern of pale areas on a dark background of small cells.(18,19) In the PC, the growth of tumor cells is believed to be favored by advantageous T-cell help, and the circulating tumor cells are the offspring of the proliferative compartment.(19) Although several previous studies have revealed that the PC play an important role in the pathogenesis of CLL/SLL,(11,19,20) the cell cycle in the PC microenvironment has not been well analyzed.(21)
In the present study, we sought to clarify the cell cycle regulation of CLL/SLL, with special reference to the PC, in terms of the expression of cyclins D2 and D3, and the molecular mechanisms involved.
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Although cyclins D2 and D3 reportedly lack a specific relationship with certain subtypes of lymphoma,(10) we found that cyclin D2 was specifically overexpressed in the PC of all CLL/SLL cases examined (19 of 19 cases). To the best of our knowledge, this is the first report that has revealed the specific overexpression of cyclin D2 using immunohistochemistry. Cyclin D3 was expressed only in a minority of tumor cells in the PC, and CLL/SLL is typically immunohistochemically negative for cyclin D1,(25) except for a few rare cases.(26) These data indicate that the cell cycle of CLL/SLL is mainly controlled by cyclin D2, supporting the hypothesis that CLL/SLL is derived from CD5-positive B1 cells,(11) because cyclin D2 plays a crucial role in mediating proliferative signaling and is essential for CD5-positive B cell development.(27)
The CCND2 gene is located on chromosome 12p13, and 14% of CLL/SLL patients have a trisomy of chromosome 12.(28) However, only a single case report has shown a translocation involving CCND2,(29) and the amplification of this gene has not been detected.
Recent studies have demonstrated that cyclin D2 expression is controlled by multiple signaling pathways, such as the NF-κB-related pathways (Fig. 3).(30) High NF-κB activity is a major finding of CLL/SLL, as well as in other B cell malignancies.(31,32) In the present study, we also identified activation of NF-κB in the PC of CLL/SLL (five of six cases). Nuclear factor (NK)-κB upregulates cyclin D2 not only by binding the promoter region of CCND2,(33) but also through c-Myc,(34,35) which also upregulates cyclin D2 expression.(36,37) Although we were not able to demonstrate the upregulation of c-Myc in the PC in the present study, it has been reported that c-Myc is upregulated in peripheral blood CLL cells activated by dipeptidyl peptidase 2 inhibition.(38)
Figure 3. The nuclear factor (NF)-κB/cyclin D2 pathway and cyclin D2 cell cycle regulation in chronic lymphocytic leukemia/small lymphocytic lymphoma. PM, plasma membrane; NM, nuclear membrane; BCR, B cell antigen receptor; PI3K, phosphatidylinositol 3-kinase; CDK, cyclin-dependent kinase; pRb, retinoblastoma protein; E2F, E2F transcription factor; P, phosphate group.
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The Ink4 family (p15, p16, p18, and p19) proteins bind to CDK4 or CDK6 and prevent interactions with their cognate D cyclins;(39) p27 (Kip1) facilitates the assembly of D cyclins and CDK4,(40) which is the primary CDK in the cell cycle of CLL/SLL (Fig. 3).(16) Although there were no significant findings for p16 and p18 in the present study, p15 was rather overexpressed in the PC examined (five of six cases); in contrast, p27 was downregulated in the PC of all CLL/SLL cases (six of six cases), in agreement with previous studies.(21) This discrepancy suggests that unknown cell cycle regulatory mechanisms involving NF-κB-related pathways exist, because c-Myc is believed to downregulate both p15 and p27.(36,41)
The proportion of mutated variants in CLL/SLL is higher in Japan than in Western countries.(14,42) In the present study, most cases (88.2%) were negative for ZAP-70. Cyclin D2 overexpression occurred in the PC of CLL/SLL samples, irrespective of ZAP-70 expression; this is in agreement with a previous study that used peripheral blood and reported that cyclin D2 can be induced in circulating CLL cells by B cell antigen receptor (BCR) stimulation, irrespective of ZAP-70 expression.(43)
It is known that ZAP-70 and CD38 are the most reliable negative prognostic markers for CLL/SLL.(44,45) Several lines of evidence imply that ZAP-70 and CD38 are not only prognostic markers, but also key elements in the pathogenesis of CLL/SLL.(46) For example, ZAP-70 can enhance and prolong activation of Syk kinase and modulate BCR signaling(47,48) and CD38 ligation induces tyrosine phosphorylation of ZAP-70, further sustaining the signal (Fig. 3).(49) These findings imply that signaling pathways regulating cyclin D2 expression can differ depending on ZAP-70 and CD38 expression. Unfortunately, in the present study the six cases whose cell cycle regulation was further analyzed were ZAP-70 negative and data for CD38 expression were only available for one of the six cases. Further investigations are required.
In conclusion, cyclin D2 is overexpressed in the PC of CLL/SLL and this overexpression is due, in part, to the upregulation of NF-κB-related pathways. These findings may play an important role in understanding cell cycle regulation in CLL/SLL because the importance of the PC is being recognized now more than before.(11,19,20)