Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program

Abstract CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments

Proportion of the CD19-Positive and CD19-Negative Lymphocytes and Monocytes within the Peripheral Blood Mononuclear Cell Set Is Characteristic for Rheumatoid Arthritis

Background and objectives: Composition of the peripheral blood (PB) cell populations and their activation state reflect the immune status of a patient. Rheumatoid arthritis (RA) is characterized by abnormal B- and T-cell functions. The objective of this study was to assess the profiles of the PB mononuclear cell (PBMC) populations in patients with rheumatoid and osteoarthritis (OA) in comparison with healthy control (HC) subjects in order to evaluate the PBMC profiles as a potential diagnostic characteristic in RA. The second aim was to assess the CCR1 and CCR2 expression on PB lymphocytes and correlate it with the plasma levels of matrix metallopeptidase 9 (MMP-9), IL-17F, TNF-α, IL-6, and IL-10. Materials and Methods: The frequency and phenotype, including CCR1 and CCR2, of the PBMC populations (monocytes, CD19+B cells, and T/NK lymphocytes) in RA (n = 15) and OA (n = 10) patients and HC (n = 12) were analyzed by five-color flow cytometry. DNA of the viruses, HHV-6, HHV-7, and B19, in the whole blood and cell-free plasma, were assessed by nested-polymerase chain reaction (PCR). Results: Active persistent or acute infections, caused by HHV-6, HHV-7, or B19, were not detected in patients of this study. Both CCR1 and CCR2 were determined on the PB B and T/NK lymphocytes in several RA and OA patients and HCs. However, in patients, the frequency of the CCR1-positive T/NK lymphocytes showed a weak negative correlation with the IL-10 level, while the frequency of the CCR2-positive B cells correlated positively with the level of IL-6. Statistically significant differences in the proportions of the CD19-positive and CD19-negative lymphocyte and monocyte subsets within the PBMC set were determined between RA and OA patients and HC adults. Conclusions: We have shown in our pilot study with rather small cohorts of patients that the PBMC-population profiles were very consistent, and statistically significantly differed between RA and OA patients and HC subjects

Chemokine Receptors CCR1 and CCR2 on Peripheral Blood Mononuclear Cells of Newly Diagnosed Patients with the CD38-Positive Chronic Lymphocytic Leukemia

Chemokines and their receptors direct migration and infiltration of immune cells. CCR1 and CCR2 maintain sequence similarity and respond to a number of the same chemokines secreted in lymphoid organs. Expression of CD38 on leukemic cells has been associated with poor clinical outcomes in patients with chronic lymphocytic leukemia (CLL) and is considered as the negative predictor of progression. In our study of newly diagnosed CLL patients, which included 39 CD38-positive and 22 CD38-negative patients, CCR1 and/or CCR2 were always detected, using flow cytometry, on the peripheral blood (PB) CD19+CD5+ lymphocytes in patients with >30% of the CD38+ CD19+CD5+ lymphocytes (n = 16). Spearman’s rank correlation analysis determined correlations between the frequency of the CCR1- and CCR2-expressing PB CD19+CD5+ lymphocytes and the frequency of the CD38-positive CD19+CD5+ lymphocytes (rs = 0.50 and rs = 0.38, respectively). No significant correlations were observed between ZAP70 mRNA expression levels in PB mononuclear cells and the frequency of the circulating CCR1+ or CCR2+ CD19+CD5+ lymphocytes. Further association studies are needed to verify prognostic relevance of the CCR1/CCR2 expression on leukemic cells in CLL patients at diagnosis. We suggest that CCR1/CCR2 signaling pathways could represent attractive targets for development of CLL anti-progression therapeutics

Mandatory chromosomal segment balance in aneuploid tumor cells

Copyright: Copyright 2013 Elsevier B.V., All rights reserved.Background: Euploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost. Methods: Fluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids. Results: In both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region. Conclusion: The inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26-q27 is consistent with the fact that the former is often deleted in human tumors, whereas the latter is frequently amplified. The findings emphasize the importance of even minor changes in copy number in seemingly unbalanced aneuploid tumors.publishersversionPeer reviewe

Positioning Europe for the EPITRANSCRIPTOMICS challenge

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.SCOPUS: no.jinfo:eu-repo/semantics/publishe

Proportion of the CD19-Positive and CD19-Negative Lymphocytes and Monocytes within the Peripheral Blood Mononuclear Cell Set Is Characteristic for Rheumatoid Arthritis

Background and objectives: Composition of the peripheral blood (PB) cell populations and their activation state reflect the immune status of a patient. Rheumatoid arthritis (RA) is characterized by abnormal B- and T-cell functions. The objective of this study was to assess the profiles of the PB mononuclear cell (PBMC) populations in patients with rheumatoid and osteoarthritis (OA) in comparison with healthy control (HC) subjects in order to evaluate the PBMC profiles as a potential diagnostic characteristic in RA. The second aim was to assess the CCR1 and CCR2 expression on PB lymphocytes and correlate it with the plasma levels of matrix metallopeptidase 9 (MMP-9), IL-17F, TNF-α, IL-6, and IL-10. Materials and Methods: The frequency and phenotype, including CCR1 and CCR2, of the PBMC populations (monocytes, CD19+B cells, and T/NK lymphocytes) in RA (n = 15) and OA (n = 10) patients and HC (n = 12) were analyzed by five-color flow cytometry. DNA of the viruses, HHV-6, HHV-7, and B19, in the whole blood and cell-free plasma, were assessed by nested-polymerase chain reaction (PCR). Results: Active persistent or acute infections, caused by HHV-6, HHV-7, or B19, were not detected in patients of this study. Both CCR1 and CCR2 were determined on the PB B and T/NK lymphocytes in several RA and OA patients and HCs. However, in patients, the frequency of the CCR1-positive T/NK lymphocytes showed a weak negative correlation with the IL-10 level, while the frequency of the CCR2-positive B cells correlated positively with the level of IL-6. Statistically significant differences in the proportions of the CD19-positive and CD19-negative lymphocyte and monocyte subsets within the PBMC set were determined between RA and OA patients and HC adults. Conclusions: We have shown in our pilot study with rather small cohorts of patients that the PBMC-population profiles were very consistent, and statistically significantly differed between RA and OA patients and HC subjects

A microcell hybrid based elimination test to identify human chromosome 3 regions that antagonize tumor growth

Deletions at multiple sites on the short arm of chr3 have been detected in many different types of human tumors. Each of the deleted regions is expected to harbor a tumor antagonizing/suppressor gene(s) (TSG). The earlier studies showed that the tumorigenicity of malignant cells could be suppressed by introducing a single human chr3. However monochromosomal hybrids system in tumorigenicity tests suffers the frequent loss of the introduced chromosome or its parts. We have therefore chosen the identification of the regions that are regularly lost in the course of mouse tumor passage. In the first study we have shown that two monochromosomal A9 mouse fibrosarcoma microcell hybrids (MCHs) lost the introduced cytogenetically intact normal human chr3 during progressive growth in SCID mice. FISH showed only fragments translocated to mouse chromosomes. PCR analysis had revealed that markers spanning the 3p21-p24 region were regularly lost/absent in all of the 13 tumors derived from 5 MCHs. We suggested that our findings might be related to the postulated presence of TSG(s) in the 3p21-p24 as indicated by the frequent deletion of this region in solid tumors. We propose that the identification of tumor antagonizing genes may be supplemented by a microcell hybrids based approach, referred as the Elimination test (Et). In the following studies, by increasing the number of tumors (27 from 5 MCHs) and chr3 markers (53), we have identified a common eliminated region (CER) of about 7 cM at 3p21.3, between D3S1260 and D3S643/D3F15SF, and a frequently eliminated region (FER) at 3p14.2-p21, between D3S1235 and D3S1067, that was absent in 21 of the 27 tumors. Using a panel of 30 SCID tumors derived from 5 MCHs that carried chr3 from the same donor, we have defined within CER at it centromeric border, at 3p21.33 between D3S1029 and LRRC2 gene, a common eliminated region 1 (CER1) of about 2.4 Mb. A common eliminated region 2 (CER2) of about 1.1 Mb was mapped within CER at it telomeric border at 3p22 between RH94338 and SHGC-154057. Unlimited availability of tumor material and the utility of non-polymorphic DNA markers in high-resolution deletion mapping are important advantages of Et. Subsequently, we have created human-human MCHs by transferring a normal human chr3 by microcell fusion into KH39 RCC cells that contained uniparentally disomic chr3. In SCID tumors derived from four of these human-human MCHs, we could identify the same eliminated regions on 3p (hCER1 and hFER) as in the human-murine model, but the centromeric border of hCER1 was shifted to the CCR2 gene away from the LRRC2 gene in human-murine CER1. Later, from the one sub-clone of the previously studied and two new A9-based mono-chromosomal MCHs that carried cytogenetically normal human chr3 from three different donors, we generated 9 SCID mice tumors that remained positive for all of the 120 3p-specific PCR-markers tested ("chr3+" tumors). These tumors were analyzed by FISH chromosome painting and by RT-PCR for the expression of 14 human chr3p genes: 5 from CER1 (LIMD1, CCR1, CCR2, CCR3, CCR5), 5 from regions that are often homozygously deleted (HD) in carcinomas (ITGA4L, LUCA1, PTPRG, FHIT, DUTT1), and 4 other cancer-related genes (VHL, MLH1, TGM4, UBE1L). Alone among the 14 genes examined, FHIT showed a tumor growth-associated change. It was expressed in MCH lines in vitro, but all of the 9 chr3+ tumors analyzed lost the FHIT gene transcript. Further we have examined the human-murine "chr3+" and human-human SCID tumors by RT-PCR for the expression of 20 chr3p genes including 7 genes from CER1 at 3p21.33 and 9 candidate TSGs from the LUCA region at 3p21.31 defined by overlapping HDs in lung cancer cell lines. We have found that in addition to the FHIT gene, the LTF gene from CER1 has also lost its mRNA expression in chr3+ tumors. Moreover, SCL38A3 from the LUCA region and DRR1 at 3p21-14.3, have down-regulated, reduced or lost, their expression after SCID passage. In the human-human system the endogenous/exogenous FHIT transcript was maintained in the SCID tumors, but the level of the Fhit protein was reduced. In contrast to the loss of mRNA expression from human-murine MCHs during SCID passage, in the human-human system, three out of the 20 examined genes, LTF, LRRC2 (neighboring LTF), and SCL38A3 (from LUCA region), expressed in the donor mouse A9-based MCH line, were suppressed, with regard to mRNA expression, after the transfer of chr3 into recipient human tumor cell line KH39. DRR1 mRNA expression was reduced in all of the 6 analyzed tumors after SCID growth. At least four of the 20 examined chr3p genes, FHIT, LTF, SCL38A3, and DRR1, showed in common a tumor-associated impairment: in human-murine system the loss or down-regulation of mRNA expression after growth in SCID mice and in human-human system the down-regulation of the expression (FHIT and DRR1) in tumors or loss of the expression (LTF and SCL38A3) already after the transfer of chr3 into the human tumor cells

Expression of the Chemokine Receptor CCR1 in Burkitt Lymphoma Cell Lines Is Linked to the CD10-Negative Cell Phenotype and Co-Expression of the EBV Latent Genes EBNA2, LMP1, and LMP2

Chemokines and their receptors regulate the migration of immune cells and the dissemination of cancer cells. CCR1, CCR2, CCR3, and CCR5 all belong to a single protein homology cluster and respond to the same inflammatory chemokines. We previously reported that CCR1 and CCR2B are induced upon Epstein-Barr virus (EBV) infection of B cells in vitro. EBV is present in almost all cases of endemic Burkitt lymphoma (BL); however, the contribution of EBV in the pathogenesis of the disease is not fully understood. Here, we analyzed the relation of the expression of CCR1, CCR2, CCR3, and CCR5, the EBV DNA load and expression of EBV latent genes in nine EBV-carrying and four EBV-negative BL cell lines. We revealed that CCR1 is expressed at high mRNA and protein levels in two CD10-negative BL cell lines with co-expression of the EBV latent genes EBNA2, LMP1, and LMP2. Low levels of CCR2 transcripts were found in three BL cell lines. CCR3 and CCR5 transcripts were hardly detectable. Our data suggest that in vivo, CCR1 may be involved in the dissemination of BL cells and in the selection of BL cells with restricted EBV gene expression programs

Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21,1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8/9 derived tumors. The third, exogenous copy of the 3q26-q27 region (part of CRR) was retained in 16/20 tumors. It can be concluded that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human/murine MCH-based test.This work was supported by grants from the Swedish Cancer Society, the Cancer Research Institute/Concern Foundation, and Karolinska Institut