Bhavana J. Dave

IL-8 Directly Enhanced Endothelial Cell Survival, Proliferation, and Matrix Metalloproteinases Production and Regulated Angiogenesis

IL-8, a member of the chemokine family, has been shown to play an important role in tumor growth, angiogenesis, and metastasis. The objective of this study was to determine the mechanism of IL-8-mediated angiogenesis. We examined the direct role of IL-8 in angiogenesis by examining IL-8 receptor expression on endothelial cells and their proliferation, survival, and matrix metalloproteinases (MMPs) production. We demonstrate that HUVEC and human dermal microvascular endothelial cells constitutively express CXCR1 and CXCR2 mRNA and protein. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube organization while neutralization of IL-8 by anti-IL-8 Ab blocks IL-8-mediated capillary tube organization. Incubation of endothelial cells with IL-8 inhibited endothelial cell apoptosis and enhanced antiapoptotic gene expression. Endothelial cells incubated with IL-8 had higher levels of Bcl-xL:Bcl-xS and Bcl-2:Bax ratios. Furthermore, incubation of endothelial cells with IL-8 up-regulated MMP-2 and MMP-9 production and mRNA expression. Our data suggest that IL-8 directly enhanced endothelial cell proliferation, survival, and MMP expression in CXCR1- and CXCR2-expressing endothelial cells and regulated angiogenesis.

BCL2 Translocation Defines a Unique Tumor Subset within the Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed prognostically important subgroups: germinal center B-cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal large B-cell lymphoma. The t(14;18)(q32;q21) has been reported previously to define a unique subset within the GCB-DLBCL. We evaluated for the translocation in 141 cases of DLBCL that were successfully gene expression profiled. Using a dual-probe fluorescence in situ hybridization assay, we detected the t(14;18) in 17% of DLBCLs and in 34% of the GCB subgroup which contained the vast majority of positive cases. In addition, 12 t(14;18)-positive cases detected by polymerase chain reaction assays on additional samples were added to the fluorescence in situ hybridization-positive cases for subsequent analysis. Immunohistochemical data indicated that BCL2, BCL6, and CD10 protein were preferentially expressed in the t(14;18)-positive cases as compared to t(14;18)-negative cases. Within the GCB subgroup, the expression of BCL2 and CD10, but not BCL6, differed significantly between cases with or without the t(14;18): 88% versus 24% for BCL2 and 72% versus 32% for CD10, respectively. In the GCB-DLBCL subgroup, a heterogeneous group of genes is overexpressed in the t(14;18)-positive subset, among which BCL2 is a significant discriminator. Interestingly, the t(14;18)-negative subset is dominated by overexpression of cell cycle-associated genes, indicating that these tumors are significantly more proliferative, suggesting distinctive pathogenetic mechanisms. However, despite this higher proliferative activity, there was no significant difference in overall or failure-free survival between the t(14;18)-positive and -negative subsets within the GCB subgroup.

BCL2 Expression Is a Prognostic Marker for the Activated B-Cell–Like Type of Diffuse Large B-Cell Lymphoma

BACKGROUND The role of BCL2 as a predictor of survival in diffuse large B-cell lymphoma (DLBCL) is controversial. DLBCL is heterogeneous, and the expression of BCL2 is variable within the two major subgroups of DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, as well as primary mediastinal DLBCL. PATIENTS AND METHODS In this study, we investigated the correlation of BCL2 expression with survival in the two major subgroups of DLBCL, as well as the mechanisms of BCL2 expression. RESULTS There was no significant correlation between BCL2 protein expression and overall survival within the GCB subgroup, but BCL2 expression had a significant adverse effect on overall survival within the ABC subgroup (P = .008). This correlation was also observed at the mRNA level (P < .04). The difference remained significant when the analyses were performed at different cutoff values. The t(14;18) was frequently observed in the GCB subgroup and was highly associated with BCL2 expression. Patients with ABC DLBCL did not exhibit t(14;18) but had a markedly higher frequency of chromosome 18q21 amplification, on which BCL2 resides. Thus, alternative mechanisms such as 18q21 amplification or activation of the nuclear factor-kappa B pathway, as reported previously, seem to be mainly responsible for the upregulation of BCL2 expression in the ABC subgroup. CONCLUSION Treating all DLBCL as a single entity ignores the mechanistic differences in BCL2 upregulation and obscures the prognostic significance of BCL2 expression. Hence, the significance of BCL2 and other biomarkers should be assessed in the context of DLBCL subgroups in future studies.

Autocrine role of interleukin-8 in induction of endothelial cell proliferation, survival, migration and MMP-2 production and angiogenesis.

Interleukin-8 (IL-8/CXCL8), a paracrine angiogenic factor, modulates multiple biologic functions in CXCR1 and CXCR2 expressing endothelial cells. Several reports suggest that inflammation, infection, cellular stress and tumor presence regulate IL-8 production in endothelial cells. In the present study, we test the hypothesis that IL-8 regulates multiple biological effects in endothelial cells in an autocrine manner. We examined the autocrine role of IL-8 in regulating angiogenesis by using a neutralizing antibody to IL-8, CXCR1 or CXCR2 in human vein umbilical endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMEC). Neutralizing antibody to IL-8, CXCR1 or CXCR2 inhibited endothelial cell proliferation, and MMP-2 production as compared to cells cultured with medium alone or control antibody. In addition, we observed that the number of apoptotic cells was significantly higher in anti-IL-8, anti-CXCR1 and anti-CXCR2 treated endothelial cells, which coincided with decreased survival-associated gene expression. We observed reduced migration of endothelial cells treated with anti-IL-8 and anti-CXCR2 antibody, but not anti-CXCR1 antibody as compared to controls. Further, we observed an inhibition of capillary tube formation and neovascularization following treatment with anti-IL-8, anti-CXCR1 and anti-CXCR2 antibodies. Together these data suggest that IL-8 functions as an important autocrine growth and angiogenic factor in regulating multiple biological activities in endothelial cells.

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

Mantle Cell Lymphoma. A Clinicopathologic Study of 68 Cases From the Nebraska Lymphoma Study Group

Although mantle cell lymphoma (MCL) is considered a distinctive disease entity within non‐Hodgkins lymphoma (NHL), the cytology and growth pattern of MCL can be quite variable and the clinical significance of these features is unclear. Also, the role of anthracyclines in the management of MCL is unclear. Therefore, we examined our experience with MCL in an effort to clarify these important issues. We identified 68 patients with MCL who were evaluated clinically and treated by the Nebraska Lymphoma Study Group. Treatment consisted of combination chemotherapy containing an anthracycline in 76% of the patients. The cases were grouped by blastic or lymphocytic cytology, and the latter were divided by growth pattern into nodular (or mantle‐zone) and diffuse types. The clinical and pathological variables were then evaluated for their prognostic value. The median overall survival (OS) and failure‐free survival (FFS) for the entire group were 38 months and 12 months, respectively, and there was no survival advantage for those who received an anthracycline. The cases were grouped as follows: blastic type, 26%; nodular lymphocytic type, 44%; and diffuse lymphocytic type, 30%. Both the cytology and pattern of growth were predictive of OS and FFS. The median OS was as follows: blastic type, 55 months; nodular lymphocytic type, 50 months; and diffuse lymphocytic type, 16 months (P = 0.0038). The clinical features that predicted for a shorter survival included bone marrow involvement, advanced stage disease, B symptoms, a poor performance score, and the International Prognostic Index. We conclude that new therapeutic approaches, with the patients stratified by histologic type and clinical prognostic factors, are clearly needed for MCL. Am. J. Hematol. 64:190–196, 2000.

Expression of CXCR1 and CXCR2 receptors in malignant melanoma with different metastatic potential and their role in interleukin-8 (CXCL-8)-mediated modulation of metastatic phenotype

In the present study, we examined the autocrine/paracrine role of IL-8 in melanoma growth and metastasis by analyzing the expression and functional significance of IL-8 receptors, CXCR1 and CXCR2 in human malignant melanoma cells with different metastatic potential. CXCR1 and CXCR2 mRNA and protein levels were analyzed by reverse trannscriptase-based polymerase chain reaction, immunohistochemistry, immunoprecipitation, flow cytometry and ligand binding assay in melanoma cells in vitro and xenografted in nude mice. Melanoma cells constitutively expressed CXCR1 and CXCR2 mRNA and protein. Highly metastatic A375SM cells expressed higher levels of CXCR1 and CXCR2 mRNA and protein in vitro and in vivo as compared to low metastatic A375P and non-metastatic SBC-2 melanoma cells. Treatment of SBC-2 and A375P cells with exogenously added recombinant IL-8 significantly enhanced their proliferation and invasive potential. Further neutralizing antibodies to CXCR1 and CXCR2 inhibited proliferation and invasive potential of unstimulated and IL-8-stimulated A375P cells. In summary, the data suggest that constitutive expression of CXCR1 and CXCR2 play an important role regulating the IL-8-mediated metastatic phenotype in human malignant melanoma cells.

Cytogenetic abnormalities and clinical correlations in peripheral T‐cell lymphoma

Cytogenetic correlations among most types of peripheral T‐cell lymphoma (PTCL) have not been very informative to date. This study aimed to identify recurrent chromosomal abnormalities in angioimmunoblastic T‐cell lymphoma (AITL), ALK‐negative anaplastic large cell lymphoma (ALK‐ALCL) and peripheral T‐cell lymphoma, unspecified (PTCL‐US), and to evaluate their prognostic value. We reviewed the cytogenetic findings of 90 previously‐diagnosed cases of PTCL and correlated the cytogenetic findings with the specific histological subtype. The most common abnormalities for AITL were 5q (55%), 21 (41%) and 3q (36%) gains, concurrent trisomies of 5 and 21 (41%), and loss of 6q (23%). In ALK(‐) ALCL, gains of 1q (50%) and 3p (30%), and losses of 16pter (50%), 6q13q21 (30%), 15 (30%), 16qter (30%) and 17p13 (30%) were frequent findings. In PTCL‐US, frequent gains involved 7q22q31 (33%), 1q (24%), 3p (20%), 5p (20%), and 8q24qter (22%), and losses of 6q22q24 (26%) and 10p13pter (26%). We did not observe any association between specific chromosomal abnormalities and overall survival (OS). However, cases with complex karyotypes, most frequently observed in ALK(‐) ALCL and PTCL‐US, had a significantly shorter OS. Although, genetic differences were noted in these subtypes, further studies are needed to determine the key pathogenetic events in PTCL.

Biotinylation of Histones Represses Transposable Elements in Human and Mouse Cells and Cell Lines and in Drosophila melanogaster

Transposable elements such as long terminal repeats (LTR) constitute approximately 45% of the human genome; transposition events impair genome stability. Fifty-four promoter-active retrotransposons have been identified in humans. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, preventing transposition events, and abnormal regulation of genes. Here, we demonstrate that the covalent binding of the vitamin biotin to lysine-12 in histone H4 (H4K12bio) and lysine-9 in histone H2A (H2AK9bio), mediated by holocarboxylase synthetase (HCS), is an epigenetic mechanism to repress retrotransposon transcription in human and mouse cell lines and in primary cells from a human supplementation study. Abundance of H4K12bio and H2AK9bio at intact retrotransposons and a solitary LTR depended on biotin supply and HCS activity and was inversely linked with the abundance of LTR transcripts. Knockdown of HCS in Drosophila melanogaster enhances retrotransposition in the germline. Importantly, we demonstrated that depletion of H4K12bio and H2AK9bio in biotin-deficient cells correlates with increased production of viral particles and transposition events and ultimately decreases chromosomal stability. Collectively, this study reveals a novel diet-dependent epigenetic mechanism that could affect cancer risk.

Genome-wide copy-number analyses reveal genomic abnormalities involved in transformation of follicular lymphoma

Follicular lymphoma (FL), the second most common type of non-Hodgkin lymphoma in the western world, is characterized by the t(14;18) translocation, which is present in up to 90% of cases. We studied 277 lymphoma samples (198 FL and 79 transformed FL [tFL]) using a single-nucleotide polymorphism array to identify the secondary chromosomal abnormalities that drive the development of FL and its transformation to diffuse large B-cell lymphoma. Common recurrent chromosomal abnormalities in FL included gains of 2, 5, 7, 6p, 8, 12, 17q, 18, 21, and X and losses on 6q and 17p. We also observed many frequent small abnormalities, including losses of 1p36.33-p36.31, 6q23.3-q24.1, and 10q23.1-q25.1 and gains of 2p16.1-p15, 8q24.13-q24.3, and 12q12-q13.13, and identified candidate genes that may be driving this selection. Recurrent abnormalities more frequent in tFL samples included gains of 3q27.3-q28 and chromosome 11 and losses of 9p21.3 and 15q. Four abnormalities, gain of X or Xp and losses of 6q23.2-24.1 or 6q13-15, predicted overall survival. Abnormalities associated with transformation of the disease likely impair immune surveillance, activate the nuclear factor-κB pathway, and deregulate p53 and B-cell transcription factors.