Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma

Abstract

The genetic events that lead to aggressive transformation of cases of splenic marginal zone lymphoma (SMZL) after the chronic clinical stage have not been well understood. We aimed to find candidate genes associated with aggressive features of SMZL. We have successfully established two SMZL cell lines, designated SL-15 and SL-22, derived from the same patient’s tumor clone in chronic and aggressive phases, respectively. Microarray analysis identified cell cycle-associated genes—specifically PLK1—as the most significantly upregulated in primary aggressive SMZL cells compared with cells from chronic phase. EPHA4 and MS4A1 (CD20) were found to be downregulated dramatically. These gene expression patterns were reproduced in both cell lines. Genetic knockdown of PLK1 resulted in inhibition of cell proliferation and induction of apoptosis in SL-22 cells, which expressed higher levels of PLK1 than SL-15 cells. SL-22 cells needed higher concentrations of chemical PLK1 inhibitors to achieve greater effects. In addition, we found homozygous deletion of the MS4A1 gene as a newly identified molecular mechanism of CD20-negative conversion. Our findings are expected to stimulate further studies on whether PLK1 could be a potential therapeutic target for this tumor. Furthermore, cases with CD20-negatively converted lymphomas should be screened for the genomic loss of MS4A1.

Prognostic significance of human papillomavirus 16 viral load level in patients with oropharyngeal cancer

“…An aliquot of cDNA was subjected to real‐time semiquantitative RT‐PCR using Power SYBR Green Master Mix (Thermo Fisher Scientific), as previously described. 22 The primers were designed to amplify the HPV16 E6 * II and E6 * I transcripts. 23 Relative HPV16 E6 mRNA expression level was calculated using the 2 −ΔCt method, with β‐actin mRNA used as a housekeeping control.…”

“…Absence of amplifiable β‐actin DNA in the extracted RNA was confirmed. An aliquot of cDNA was subjected to real‐time semiquantitative RT‐PCR using Power SYBR Green Master Mix (Thermo Fisher Scientific), as previously described 22 . The primers were designed to amplify the HPV16 E6 * II and E6 * I transcripts 23 .…”

Generation and characteristics of a novel “double-hit” high grade B-cell lymphoma cell line DH-My6 with MYC/IGH and BCL6/IGH gene arrangements and potential molecular targeted therapies

“…Based on reviews in the literature [ 14 , 33 , 34 ], most of the MYC / BCL2 DHL cases showed decreased expression of CD20. Although mechanisms resulting in decreased expression of CD20 after rituximab treatment have been studied using rituximab-resistant lymphoma cell lines and the results have suggested the association of epigenetic or genetic changes with downregulation of CD20 [ 35 , 36 ], the underlying mechanism for diminishing CD20 expression in de novo DHL cells has not been investigated. Clinically, the decreased expression of CD20 results in the loss of efficiency of anti-CD20 antibody therapies.…”

“…The reaction was conducted on a StepOnePlus thermocycler (Thermo Fisher Scientific) with THUNDRBIRD SYBR qPCR mix (TOYOBO, Osaka, Japan) containing 0.3 μM of each primer. PLK1 expression in DH-My6 cells was also examined as described previously [ 35 ]. The primer sequences used to determine the gene expressions are listed in Supplementary Table 1 .…”

“…To compare the antiproliferation effect of single-agent treatments with combination treatments, synergy levels were determined from CI ranges, using the Chou–Talalay method [ 59 ]: no synergism, CI > 1.1; additive effect, CI = 1.0–1.1; and synergism, CI < 1.0. For apoptosis analysis, cells were stained with annexin V-phycoerythrin and 7-amino-actinomycin D as described [ 35 ]. For cell cycle analysis, cells were fixed in cold 70% ethanol, treated with RNase, and stained with propidium iodide.…”