Human B-cell Lymphoma Cell Lines Research Articles

Resistance to TGF-β1 correlates with aberrant expression of TGF-β receptor II in human B-cell lymphoma cell lines

AbstractResistance to transforming growth factor (TGF)–β1–mediated growth suppression in tumor cells is often associated with the functional loss of TGF-β receptors. Here we describe two B-cell lymphoma cell lines (DB and RL) that differ in their sensitivity to TGF-β1–mediated growth suppression. The TGF-β1–resistant cell line DB lacked functional TGF-β receptor II (TβRII) in contrast to the TGF-β–responsive cell line RL, whereas both cell lines had comparable levels of receptor I (TβRI). Lack of functional TβRII was correlated with the lack of TGF-β1–induced nuclear translocation of phospho-Smad3 and phospho-Smad2, the lack of nuclear expression of p21Cip1/WAF1, and the down-regulation of c-Myc in DB cells. Transfection of wild-type, but not a C-terminal–truncated, form of TβRII rendered the DB cell line responsive to TGF-β1–mediated growth suppression. Analysis of the TβRII gene in DB cells revealed the absence of TβRII message, which was reversed upon 5′-azacytidine treatment, indicating that the promoter methylation might be the cause of gene silencing. Promoter analysis revealed CpG methylations at −25 and −140 that correlated with the gene silencing. These data suggest that promoter methylation plays an important role in TβRII gene silencing and subsequent development of a TGF-β1–resistant phenotype by some B-cell lymphoma cells.

Preclinical pharmacokinetics, pharmacodynamics and activity of a humanized anti‐CD40 antibody (SGN‐40) in rodents and non‐human primates

British Journal of Pharmacology (2007) 150, 248. doi:10.1038/sj.bjp.0707129

The Green Tea Extract Epigallocatechin Induces In Vitro Cell Death in Primary Human Lymphoma Cells through an ROS Dependent Mechanism.

BACKGROUND: We have demonstrated the green tea extract epigallocatechin-3-gallate (EGCG) has anticancer activity in primary CLL B-cells (Lee, Blood 2004). After dissemination of our in vitro findings by the lay press, many patients with CLL and other low grade non-Hodgkin lymphomas (NHL) began using over the counter green tea extracts as an alternative treatment strategy. We recently reported a case series of 3 patients with CLL and 1 patient with follicular lymphoma who appeared to derive objective clinical benefit from such treatment (Shanafelt, Leukemia Research 2006). Based on these findings EGCG has entered clinical testing for treatment of CLL at Mayo Clinic. Here we explore the in vitro antitumor activity of EGCG against other types of non-Hodgkin lymphoma.METHODS: Five established human B-cell lymphoma cell lines (HT, DOHH2, KARPAS, Ramos, RL) and primary lymphoma cells from 7 patients with various B-cell NHL sub-types [DLBC, FL, SMZ (2), MCL, SLL(2)] were used to evaluate the in vitro sensitivity of human lymphoma cells to EGCG. Freshly isolated primary lymphoma cells harvested in suspension from lymph nodes/spleen were obtained from patients with NHL who provided written informed consent. All patients were untreated at the time of biopsy. Lymphoma cell lines and primary lymphoma cells (n=7) were cultured with increasing doses of purified EGCG (3.12–50 ug/ml) for 24–72 hrs. Viability was assessed by using annexin/PI staining by FACS analysis.RESULTS: EGCG-induced dose dependent cell death in both established human B-cell lymphoma cell lines (average LD50 at 24 hrs between 25–50 ug/mL) and primary NHL cells (average LD50 at 24 hrs between 25–50ug/mL). In contrast, EGCG had minimal effect on purified normal B-cells (n=3) at the highest doses tested (50 ug/mL). By immunoblotting, EGCG-induced death in primary cells and cell lines was associated with PARP cleavage, suggesting the agent induced apoptotic cell death. Despite this finding, EGCG had no effect on levels of MCL-1, XIAP, or Bcl-1 by either immunoblot or FACS analysis. Based on reports that EGCG induces cell death in some cancer cell types through generation of oxidative stress (Furukawa, 2003; Nakazato, 2005), we explored this mechanism in lymphoma cells. To determine whether reactive oxygen species (ROS) generation was necessary for EGCG-induced cell death, lymphoma cell lines were cultured with or without catalase (which catalyzes the conversion of hydrogen peroxide to water and oxygen) for 30 min prior to subsequent 24 hr EGCG exposure (50 and 100 mg/ml). Pre-treatment with catalase (100 U) provided dramatic protection against cell death in both primary NHL cells and NHL cell lines suggesting that EGCG-induced cell death in lymphoma cells is dependent on ROS generation (Fig. 1 shows an example for a patient with mantle cell lymphoma and a patient with splenic marginal zone lymphoma).CONCLUSION: EGCG has in vitro anti-tumor activity against a variety of B-cell NHLs. Given its known favorable toxicity profile in vivo, EGCG is an attractive agent for clinical testing in patients with indolent NHL who otherwise are currently being managed with observation. [Display omitted]

Efficacy of celecoxib in the treatment of CNS lymphomas: an in vivo model

The incidence of primary central nervous system lymphomas (PCNSLs) has increased over the past several decades. Unfortunately, even with the most effective therapeutic regimen (that is, methotrexate with wholebrain radiation therapy), PCNSL recurs within a few years in more than half of the treated patients and is eventually fatal. Because PCNSL usually occurs in older patients and in those with acquired immunodeficiency syndrome, combination treatments in which both chemo- and radiation therapy are used is often poorly tolerated and results in a significant reduction in the quality of life. Recently, it has been demonstrated that the selective cyclooxygenase- 2 inhibitor celecoxib (Celebrex), can block the growth of lymphoma cells in vitro. To create an experimental animal model in vivo for the PCNSL study, the authors intracranially injected a human B-cell lymphoma cell line into nude mice. Their data demonstrate that this experimental model is an excellent one for human PCNSL with brain and leptomeningeal involvement. They also evaluated the feasibility of using celecoxib as a therapeutic agent in the treatment of PCNSL. Nude mice with intracranial lymphomas were treated with celecoxib contained in the animal chow. The treated animals demonstrated significantly prolonged survival times compared with the untreated animals. Based on the authors' data, celecoxib may be a promising therapeutic agent for the treatment of PCNSL.

Preclinical pharmacokinetics, pharmacodynamics, and activity of a humanized anti-CD40 antibody (SGN-40) in rodents and non-human primates.

1. Cell-surface expression of CD40 in B-cell malignancies and multiple solid tumors has raised interest in its potential use as a target for antibody-based cancer therapy. SGN-40, a humanized monoclonal anti-CD40 antibody, mediates antibody-dependent cytotoxicity and inhibits B-cell tumor growth in vitro, properties of interest for the treatment of cancers, and is currently in Phase I clinical trials for B-cell malignancies. In this study, we determined in vivo activity and pharmacokinetics properties of SGN-40. 2. Effect of SGN-40 in xenograft model of CD40-expressing B-cell lymphoma in severe-combined immune deficiency mice and its in vivo pharmacokinetics properties in normal mice, rats and cynomolgus monkeys were studied. 3. Treatment with SGN-40 significantly increased the survival of mice xenografted with human B-cell lymphoma cell line. SGN-40 exhibited nearly 100% bioavailability in mice and it cleared faster when given at a low dose. In monkeys, clearance of SGN-40 was also much faster at low dose, suggesting nonlinear pharmacokinetics in these species. In rats, however, SGN-40 clearance at all tested doses was similar, suggesting that pharmacokinetics were linear in this dose range in rats. Administration of SGN-40 to monkeys also produced marked, dose-dependent, and persistent depletion of peripheral CD20(+) B lymphocytes. 4. Data presented in this report suggest that SGN-40 is active in in vivo, and based upon interspecies scaling, SGN-40 clearance in humans is predicted to be similar to observed SGN-40 clearance in monkeys. These data suggest that SGN-40 has appropriate pharmacokinetic properties that support its clinical use.

Motexafin Gadolinium and Zinc Induce Oxidative Stress Responses and Apoptosis in B-Cell Lymphoma Lines

There is an emerging appreciation of the importance of zinc in regulating cancer cell growth and proliferation. Recently, we showed that the anticancer agent motexafin gadolinium (MGd) disrupted zinc metabolism in A549 lung cancer cells, leading, in the presence of exogenous zinc, to cell death. Here, we report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in exponential phase human B-cell lymphoma and other hematologic cell lines. We find that increased levels of oxidative stress and intracellular free zinc precede and correlate with cell cycle arrest and apoptosis. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1)-regulated and hypoxia-inducible transcription factor-1 (HIF-1)-regulated genes. Cultures cotreated with MGd and zinc acetate displayed further increases in the levels of MTF-1- and HIF-1-regulated transcripts as well as additional transcripts regulated by NF-E2-related transcription factor 2. These data provide insights into the molecular changes that accompany the disruption of intracellular zinc homeostasis and support a role for MGd in treatment of B-cell hematologic malignancies.

Motexafin Gadolinium Causes Apoptosis in Lymphoma Cells by Increasing Intracellular Free Zinc and Disrupting Redox Balance.

Abstract Motexafin gadolinium (MGd, Xcytrin®) is an anti-cancer agent that is being studied in clinical trials in various types of cancer including hematologic malignancies. MGd has been shown to generate reactive oxygen species and induce apoptosis in myeloma and lymphoma cells, and to cause an increase in intracellular levels of free zinc. We now report the effect of MGd and exogenous zinc on intracellular levels of free zinc, oxidative stress, proliferation, and cell death in human B-cell lymphoma and other hematologic cell lines. To better understand the molecular basis of these cellular responses, gene expression profiling analyses were conducted on Ramos cell cultures treated with MGd and/or zinc acetate. In cultures treated with MGd and zinc, 21% of Ramos cells were apoptotic within 8 hours of treatment as demonstrated by Annexin V staining. The apoptotic fraction increased to 30% within 12 hours and 68% by 24 hours. Analogous results were obtained using JC-1, with 38% of Ramos cells exhibiting non-aggregated (green) JC-1 fluorescence characteristic of mitochondrial dysfunction within 8 hours of combined treatment with MGd and zinc. This fraction increased to 52% by 12 hours and 74% by 24 hours. MGd and zinc similarly increased cell death in other B-cell lines (Raji, DB, DHL-4 and HF-1) tested. We also found that increased levels of oxidative stress and intracellular free zinc preceded and correlated with cell cycle arrest and apoptosis. Cultures treated with MGd or zinc acetate alone elicited transcriptional responses characterized by induction of metal response element-binding transcription factor-1 (MTF-1) and hypoxia inducible transcription factor 1 (HIF-1) regulated genes. Cultures co-treated with MGd and zinc acetate displayed further increases in the levels of MTF-1 and HIF-1 regulated transcripts and also additional transcripts regulated by NF-E2-related transcription factor 2 (NRF-2). HIF-1 activation can have negative consequences for cell growth and survival by induction of targets linked to apoptosis, whereas NRF-2 activation is characteristic of cells responding to disruption of redox balance. These data demonstrate that MGd increases intracellular free zinc and oxidative stress levels in lymphoma cells that activate adaptive survival responses but eventually lead to cell death.

Novel follicular dendritic cell molecule, 8D6, collaborates with CD44 in supporting lymphomagenesis by a Burkitt lymphoma cell line, L3055

AbstractThe lymphoid follicle is a specialized microenvironment for the differentiation of antigen (Ag)–activated B cells; the major stromal cell components in lymphoid follicle are the follicular dendritic cells (FDCs). At the same time, most of the B-cell lymphomas originate from the germinal center, and the generation and blast transformation of B-cell lymphoma occurs in close association with FDCs in the early stage of tumorigenesis. To study the functional roles of FDCs in lymphomagenesis, we established an inducible tumor model. The human B-cell lymphoma cell line, L3055, formed solid tumors only when inoculated with an FDC line, HK. In addition, 2 FDC-signaling molecules (FDC-SMs), a novel protein 8D6 and 4G10/CD44, are required for tumor formation in vivo, because monoclonal antibodies (mAbs) specific to these 2 proteins inhibited lymphomagenesis completely when they were inoculated with L3055 and HK cells. However, these 2 FDC-SMs have distinct functional roles in tumor formation. FDC-SM-8D6 enhances L3055 cell proliferation, whereas FDC-SM-4G10/CD44 inhibits its apoptosis. Identification of the functional roles of these critical FDC-SMs may lead to the discovery of therapeutic drugs that suppress the survival and growth of lymphoma cells.

Fenretinide enhances rituximab-induced cytotoxicity against B-cell lymphoma xenografts through a caspase-dependent mechanism

AbstractThe anti-CD20 monoclonal antibody rituximab induces remission in 40% to 60% of patients with indolent B-cell lymphoma, but virtually all patients have relapses. We evaluated the efficacy of concurrent administration of another biologic agent, N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) with rituximab against a variety of human B-cell lymphoma cell lines (Ramos, DHL-4, and FL-18) in vivo. Concurrent 4HPR and rituximab administration prevented tumor progression of lymphoma-bearing mice in a minimal disease model (rituximab + 4HPR, 100% progression free; rituximab alone, 37.5% progression free, P = .01; 4HPR alone, 12.5% progression free, P < .01; controls, 0% progression free, P < .01). Combinations of 4HPR + rituximab exceeded the predicted 50% additive rate of disease control from each agent alone (P = .038). Administering 4HPR and rituximab to mice with established tumors induced complete responses (CRs) in 80% of animals compared with 20% to 40% CRs using either agent alone (P = .07), resulting in significantly improved survival. Tumors harvested from 4HPR + rituximab-treated mice displayed elevated caspase activation compared with untreated controls (P = .02). Adding a broad-spectrum caspase inhibitor in vivo fully abrogated the antitumor effects of 4HPR + rituximab (P = .05). These results establish the efficacy of 4HPR/rituximab combinations, confirm their caspase-mediated mechanism of action, and offer the potential for disease control with minimal toxicity for patients with B-cell malignancies. (Blood. 2004;103:3516-3520)

Construction of heteroduplex DNA andin vitro model for functional analysis of mismatch repair

Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZα gene have been used to construct two kinds of hetero-duplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G·G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. coli NR9162 (mutS-) without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100× (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G-G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G-G is less than 20%. Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary nonpolyposis colorectal cancer (microsatellite instability high, MSI-H) was measured and lack of MMR ability was shown. And a case of sporadic rectal cancer (SRC) (microsatellite stability, MSS) maintains MMR proficiency. The results indicate that the model is sensitive and dependable. It could be used to measure the func- tion status of MMR system in tumor cell and/or tissues. This is a reliable method to investigate the mechanic of tumori-genesis. It is meaningful in the observation of the role of MMR in the initiation and progression of concerned tumors.

Crystallization and X-ray analysis of the N-terminal core domain of a tumour-associated human DEAD-box RNA helicase, rck/p54.

The RCK gene was cloned on the basis of the t(11;14)(q23;q32) chromosome translocation observed in human B-cell lymphoma cell line RC-K8. This gene was found to be overexpressed in various kinds of tumours. The gene product, rck/p54, consisting of 472 amino-acid residues with molecular weight 53.2 kDa, belongs to the family of DEAD-box RNA helicases. Its ATP-dependent RNA-unwinding activity toward c-myc RNA molecules in vitro has recently been demonstrated. In the present study, limited proteolysis experiments of rck/p54 were used to truncate the N-terminal domain (residues 1-288; 31.8 kDa) of rck/p54, leading to successful crystallization of Nc-rck/p54, i.e. the N-terminal core domain (residues 70-288; 24.5 kDa) of rck/p54. Crystals of Nc-rck/p54 were grown to a size suitable for X-ray structure analysis using polyethylene glycol 3350 as the precipitant. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 65.5, b = 73.1, c = 84.8 A, and diffracts X-rays to beyond 2.0 A resolution.

Overexpression of a DEAD box/RNA helicase protein, rck/p54, in human hepatocytes from patients with hepatitis C virus-related chronic hepatitis and its implication in hepatocellular carcinogenesis.

Hepatitis C virus (HCV) infection is the most common cause of chronic hepatitis, which frequently progresses to hepatocellular carcinoma. The pathogenesis of its persistent infection and tumour progression has not been fully characterized yet. The RCK gene was previously cloned at the breakpoint of the t(11;14)(q23;q32) chromosome translocation observed in human B-cell lymphoma cell line RC-K8. The RCK protein, rck/p54, which is a 54-kDa cytoplasmic protein belonging to the DEAD box/RNA helicase family, is considered to facilitate the translation of mRNA(s) of genes for cell proliferation and malignant transformation not only in B-cell lymphomas having the t(11;14) translocation but also in other solid tumours. The aim of this work was to examine the involvement of rck/p54 in carcinogenesis of hepatocellular carcinoma from HCV-related chronic hepatitis. We examined the expression of rck/p54 in 29 cases of HCV-related chronic hepatitis and eight cases of hepatocellular carcinoma by immunohistochemistry and Western blot analysis. Twenty-six of 29 cases with HCV-related chronic hepatitis and all cases with hepatocellular carcinoma tested overexpressed rck/p54 protein. The expression of rck/p54 was lowered by treatment with IFN-alpha in two cases who showed the decrease in HCV RNA levels. These findings suggest that rck/p54 protein is possibly involved in the replication of HCV genomes in hepatocytes and in tumourigenesis of hepatocellular carcinomas.

Reversal of Bcl-2-mediated resistance of the EW36 human B-cell lymphoma cell line to arsenite- and pesticide-induced apoptosis by PK11195, a ligand of the mitochondrial benzodiazepine receptor.

Opening of the permeability transition (PT) pore is a central feature of apoptosis induction by chemical stress. One component of the PT pore, the mitochondrial benzodiazepine receptor (mBzR), has recently received attention for its potential role in modulating PT pore function. Specifically, antagonistic ligands of the mBzR, such as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide (PK11195), have been shown to sensitize Bcl-2 overexpressing cells to apoptosis induction by facilitating the opening of the PT pore and the subsequent loss of mitochondrial membrane potential (Deltapsim). We examined whether PK11195 can sensitize EW36, a human B-cell lymphoma cell line that over-expresses Bcl-2, to apoptosis induction and mitochondrial depolarization by environmental chemicals including mitochondrial toxicants. We found that, although EW36 cells are refractory to apoptosis induction by antimycin A, rotenone, pyridaben, alachlor, and carbonyl cyanide m-chlorophenylhydrazone (mClCCP), they are dramatically sensitized to induction of apoptosis by low concentrations of these same agents following pre-treatment with PK11195. The sensitization of EW36 cells is accompanied by a rapid and extensive loss of Deltapsim within a few hours following chemical exposure. Furthermore, using sodium arsenite, we examined the role of the c-Jun N-terminal kinase (JNK) pathway and protein synthesis in apoptosis induction in EW36. We found that, unlike untreated cells, EW36 cells treated with PK11195 no longer show an association of JNK pathway activation with apoptosis induction. Importantly, PK11195 eliminates a requirement for protein synthesis in chemically induced apoptosis in EW36 cells. These results show significant drug-mediated alteration of cell sensitivity and JNK pathway activation to environmental chemicals and mitochondrial toxicants, following ligation of the mBzR.

The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-kappaB signal transduction pathway.

The human large B-cell lymphoma cell line RC-K8 has a rearranged REL locus that directs the production of a chimeric protein, termed REL-NRG (Non-Rel Gene). In this study, we show that RC-K8 cells have constitutively nuclear heterodimeric and homodimeric DNA-binding complexes that consist of p50, REL, and REL-NRG. In vitro, IkappaBalpha can block the DNA-binding activity of wild-type REL homodimers but not REL-NRG homodimers. In vivo, REL-NRG cannot activate transcription of a kappaB site reporter plasmid, suggesting that it is a transcription repressing or blocking REL protein. By Western blotting, no IkappaBalpha protein can be detected in extracts of RC-K8 cells. The absence of IkappaBalpha protein in RC-K8 cells appears to be due to mutations that cause premature termination of translation in three of the four copies of the IKBA gene in RC-K8 cells. Re-expression of wild-type IkappaBalpha or a super-repressor form of IkappaBalpha in RC-K8 cells is cytotoxic; in contrast, expression of a dominant-negative form of IkappaB kinase does not affect the growth of RC-K8 cells. By cDNA microarray analysis, a number of previously identified Rel/NF-kappaB target genes are overexpressed in RC-K8 cells, consistent with there being transcriptionally active REL complexes. Taken together, our results suggest that the growth of RC-K8 cells is dependent on the activity of nuclear wild-type REL dimers, while the contribution of REL-NRG to the transformed state of RC-K8 cells is less clear. Nevertheless, the RC-K8 cell line is the first tumor cell line identified with mutations in genes encoding multiple proteins in the Rel/NF-kappaB signal transduction pathway.

Genomic organization and expression of the rearranged REL proto‐oncogene in the human B‐cell lymphoma cell line RC‐K8

The human large B-cell lymphoma cell line RC-K8 has a rearranged REL locus that is transcribed into a chimeric mRNA, termed REL-NRG (Non-Rel Gene). By analyzing the recently completed human genome sequence, we have found that the normal REL and NRG loci are separated by approximately 28 megabase pairs on chromosome 2, suggesting that a deletion created the REL-NRG locus in RC-K8 cells. Using computer-based and molecular approaches, we have determined the structure of the altered REL locus in RC-K8 cells. The REL-NRG transcript is encoded by 7 REL exons and 6 NRG-derived exons. Direct DNA sequencing has identified the site of the REL-NRG fusion in RC-K8 cells. We also show that both wild-type c-Rel and c-Rel-Nrg proteins are expressed and in a complex in RC-K8 cells. Furthermore, like c-Rel, c-Rel-Nrg is a cytoplasmic protein when overexpressed in fibroblasts in culture and can bind to a kappaB DNA site in vitro.

Human B-cell lymphoma cell lines are highly sensitive to apoptosis induced by all- trans retinoic acid and interferon-γ

When cells were incubated in the presence of both interferon-γ (IFN-γ) and all- trans retinoic acid (ATRA), the concentration of IFN-γ required to induce apoptosis of B-cell lymphoma cells was much lower than that required for myeloid or erythroid cell lines. The concentration of IFN-γ that effectively inhibited the proliferation of BALM-3 cells was 1/40 of that required for BALM-1 cells. STAT-1 phosphorylation, IRF-1 mRNA and protein expression and RAR-β expression were enhanced to a greater degree in BALM-3 cells treated with IFN-γ and ATRA than in BALM-1 cells treated with IFN-γ and ATRA, suggesting that these IFN-γ related genes were involved in the induction of apoptosis of BALM-3 cells.

Structure of the human retinoblastoma-related p107 gene and its intragenic deletion in a B-cell lymphoma cell line

The human p107 protein shares many structural and functional features with the retinoblastoma gene product and retinoblastoma-related p130 protein. In this study, we have cloned and elucidated the complete intron–exon organization of the gene encoding the p107 protein. The gene contains 22 exons spanning over 100 kilobase pairs of genomic DNA. The length of individual exons ranges from 50 to 840 base pairs. The arrays of exons in the p107 gene are rather similar among members of the gene family, especially to those of the p130 gene, while the length of introns is extensively diverse. This study will provide a molecular basis for implementing comprehensive screening for p107 mutations using genomic DNAs from human malignancies. We also show a detailed structure of an intragenic deletion of the p107 gene found in a human B-cell lymphoma cell line, KAL-1, which was shown to occur by homologous recombination between the two directly repeated Alu family sequences.

4-Hydroxynonenal, an end-product of lipid peroxidation, induces apoptosis in human leukemic T- and B-cell lines

4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells.

Inhibition of Human Breast Carcinoma Growth by a Soluble Recombinant Human CD40 Ligand

AbstractCD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-γ. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40+ human breast cancer cell lines. This inhibition could also be augmented with interferon-γ. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.

Inhibition of Human Breast Carcinoma Growth by a Soluble Recombinant Human CD40 Ligand

CD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-γ. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40+ human breast cancer cell lines. This inhibition could also be augmented with interferon-γ. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.