Genetic alteration associated with chronic lymphocytic leukemia.

The genetics of B-cell chronic lymphocytic leukemia (B-CLL) differ considerably from most other forms of hematologic malignancy which are usually characterized by chromosome translocations. B-CLL typically contains chromosomal deletions and chromosomes 13q14 and 11q22-->q23 are the most common. These two regions appear to share a common ancestral origin (Auer et al., 2007b). Overall, chromosomal abnormalities can be found in the majority of patients with B-CLL when using sensitive techniques (Dohneret al., 2000) and possibly reflects an underlying predisposition, with a small but significant number of familial cases. Although single and consistent abnormalities are most common, multiple rearrangements can occur, often with disease progression (Feganetal., 1995; Dohner et al., 2000). Regions of recurrent deletion suggest the presence of tumor suppressor genes if following Knudson's theoretical 2-hit model. However, despite extensive sequencing analysis over the last decade and lack of pathogenic mutations identified, there has been a move away from this suggested hypothesis and alternative mechanisms of gene inactivation involving epigenetic silencing or haploinsufficiency may be considered as more likely in this disease. This review focuses on the common genetic abnormalities in B-CLL and relates them to some of the more recent hypotheses on inactivation of genes within these regions of deletion.

Stochastic modelling of apoptosis kinetics.

Robust quantitative estimation of average whole cell mitochondrial dysfunction is a useful tool for assessing sensitivity to apoptotic stimuli induced either by novel agents, or following manipulation of apoptotic threshold by pharmacological or functional genomics approaches. We have mathematically modelled the kinetics of whole cell mitochondrial membrane potential depolarisation within a population of cells as a Bernouli transition. An exponential distribution enables the median latency preceding mitochondrial membrane potential dissipation to be derived. The kinetic model can be fitted to in vitro single cell resolution data derived from kinetic flow cytometric studies by non-linear regression. We propose that kinetic determination of cumulative frequency distributions provides a useful approach for estimating apoptosis sensitivity across cell populations over short time-frames.

Pk11195, a mitochondrial benzodiazepine receptor antagonist, reduces apoptosis threshold in Bcl-X(L) and Mcl-1 expressing human cholangiocarcinoma cells.

BACKGROUND: Cholangiocarcinoma cells express high levels of the antiapoptotic proteins Bcl-X(L) and Mcl-1 and are markedly chemo- and radioresistant. Mitochondria have emerged as central players in apoptosis. Antiapoptotic members of the Bcl-2 protein family localise to the outer mitochondrial membrane and regulate mitochondrial release of apoptogenic proteins. Mitochondrial benzodiazepine receptor (mBzR) ligands have been shown to reverse Bcl-2 action and facilitate apoptosis. AIM: We evaluated the ability of the mBzR antagonist Pk11195 to overcome preapoptotic mitochondrial dysfunction in Egi-1 and Tfk-1, two human cholangiocarcinoma cell lines expressing high levels of Bcl-X(L) and Mcl-1. MATERIALS AND METHODS: Cells growing in culture were used to perform in vitro experiments over 48-96 hours following treatment. The cytotoxic agents used were 5 fluorouracil 10 microM and etoposide (Vp16) 10 microM, together with ultraviolet and 0.5-1 Gy x ray irradiation with or without 75 microM Pk11195. Apoptosis and mitochondrial dysfunction were measured at single cell resolution by flow cytometry using the mitochondrial fluorochrome DiOC6(3). Severe combined immunodeficient non-obese diabetic (SCID-NOD) mice with subcutaneous xenografts using the Egi-1 and Tfk-1 cell lines were treated with etoposide with or without addition of Pk11195 over a 72 hour period during which time the xenograft growth patterns were monitored. RESULTS: In vitro, the effect of Pk11195 on induction of apoptosis in cholangiocarcinoma cells following stimulation by chemotherapy or radiotherapy was found to be both time and dose dependent, with Pk11195 increasing rates of apoptosis by 50-95%. Intraperitoneal administration of Pk11195 in combination with Vp16 was found to increase the growth inhibiting effects of Vp16 on xenografts during the treatment phase. PK11195 75 microM on its own had no intrinsic cytotoxic efficacy. CONCLUSION: This is the first study to demonstrate that functional antagonism of coexpressed Bcl-X(L) and Mcl-1 proteins using the mBzR antagonist Pk11195 can facilitate apoptosis in cholangiocarcinoma following chemotherapy and radiotherapy.

Bcl-2 resistant mitochondrial toxicity mediated by the isoquinoline carboxamide PK11195 involves de novo generation of reactive oxygen species.

Resistance to apoptosis is a major obstacle preventing effective therapy for malignancy. Mitochondria localized anti-death proteins of the Bcl-2 family play a central role in inhibiting apoptosis and therefore present valid targets for novel therapy. The peripheral benzodiazepine receptor (PBR) shares a close physical association with the permeability transition pore complex (PTPC), a pivotal regulator of cell death located at mitochondrial contact sites. In this study we investigated the cytotoxicity of the PBR ligand, PK11195, in the micromolar concentration range. PK11195 induced antioxidant inhibitable collapse of the inner mitochondrial membrane potential (DeltaPsi(m)) and mitochondrial swelling in HL60 human leukaemia cells, but not in SUDHL4 lymphoma cells (which exhibited a higher level of reduced glutathione and relative tolerance to chemotherapy or pro-oxidant induced DeltaPsi(m)dissipation). PK11195 induced the production of hydrogen peroxide that was not inhibited by Bcl-2 transfection, nor depletion of mitochondrial DNA. ROS production was however blocked by protonophore, implicating a requirement for DeltaPsi(m). Our findings suggest that PK11195-induced cytotoxicity relies upon Bcl-2 resistant generation of oxidative stress; a process only observed at concentrations several orders of magnitude higher that required to saturate its receptor.

In vivo suppression of Bcl-XL expression facilitates chemotherapy-induced leukaemia cell death in a SCID/NOD-Hu model.

Bcl-XL, a member of the Bcl-2-related anti-apoptosis protein family, antagonizes a diverse range of apoptosis-inducing stimuli by preventing mitochondrial permeability transition, release of apoptogenic factors including cytochrome C, and caspase activation. We have tested the hypothesis that the susceptibility of Bcl-XL-expressing leukaemic cells to apoptosis induced by VP16 (etoposide) can be enhanced by pharmacological downregulation of Bcl-XL in vivo. Two subcutaneous xenograft models of B-cell leukaemia-employing SEMK-2 and BV173 cell lines were established in severe combined immunodeficient/non-obese diabetic mice followed by 14 d of continuous subcutaneous administration of Bcl-XL-specific second generation oligonucleotides ISIS 16009 or ISIS 15999. Tumours were disaggregated, enabling investigation of Bcl-XL expression and apoptosis susceptibility at single-cell resolution using cytofluorimetry. Marked sequence-specific reduction of Bcl-XL was associated with sequence-specific enhancement of VP16-induced mitochondrial permeability transition, caspase-3 activation and loss of membrane asymmetry. A negative correlation between Bcl-XL expression and apoptosis susceptibility was observed, together with a positive correlation with respect to a reduced redox state. Bcl-XL downregulation reduces the threshold for VP16-induced apoptosis by potentiating mitochondrial dysfunction and its sequelae, and therefore presents a novel therapeutic strategy for reversing chemoresistance.

A dynamical systems model to simulate the perturbation kinetics of gene expression by antisense oligonucleotides.

Antisense oligonucleotides owe their efficacy to an ability to induce RNase H-dependent suppression of RNA translation, for sufficient time to allow physiological proteolysis. The magnitude and time delay preceding the protein nadir concentration determine the extent and timing of maximum antisense oligonucleotide activity. Antisense oligonucleotide degradation underlies reversal of RNA downregulation. The kinetics of protein downregulation is therefore determined by the complex interaction of both ligand chemistry (nuclease stability, affinity and RNase H activation), and gene expression kinetics. Optimization of antisense oligonucleotide efficacy and experimental design requires understanding of these interactions. The kinetics of protein and RNA downregulation have therefore been simulated by analysing a two-compartment kinetic model incorporating RNase H-dependent transcript degradation. The system of nonlinear differential equations describing this model was solved numerically using Runge-Kutte integration. The timecourse solutions corresponding to the four state variables (RNA, protein, antisense/RNA heteroduplex and antisense oligonucleotide), were determined simultaneously. This allowed systematic in silico examination of the consequences of altering variables such as oligonucleotide concentration, affinity, and stability, or the scheduling of multiple transfections on RNA and protein perturbations. By providing a tool for examining antisense oligonucleotide action theoretically, this heuristic model should facilitate both the rational design and interpretation of antisense experiments.

Role for CCG-trinucleotide repeats in the pathogenesis of chronic lymphocytic leukemia.

Chromosome 11q deletions are frequently observed in chronic lymphocytic leukemia (CLL) in association with progressive disease and a poor prognosis. A minimal region of deletion has been assigned to 11q22-q23. Trinucleotide repeats have been associated with anticipation in disease, and evidence of anticipation has been observed in various malignancies including CLL. Loss of heterozygosity at 11q22-23 is common in a wide range of cancers, suggesting this is an unstable area prone to chromosome breakage. The location of 8 CCG-trinucleotide repeats on 11q was determined by Southern blot analysis of a 40-Mb YAC and PAC contig spanning 11q22-qter. Deletion breakpoints in CLL are found to co-localize at specific sites on 11q where CCG repeats are located. In addition, a CCG repeat has been identified within the minimal region of deletion. Specific alleles of this repeat are associated with worse prognosis. Folate-sensitive fragile sites are regions of late replication and are characterized by CCG repeats. The mechanism for chromosome deletion at 11q could be explained by a delay in replication. Described here is an association between CCG repeats and chromosome loss suggesting that in vivo "fragile sites" exist on 11q and that the instability of CCG repeats may play an important role in the pathogenesis of CLL.

Antisense Bcl-2 oligonucleotide uptake in human transitional cell carcinoma.

OBJECTIVES: Antisense oligonucleotides (AO) downregulate Bcl-2 protein expression in various tumours if good target cell uptake is achieved. In this study, uptake of FITC labelled AO (FITC-AO) directed at Bcl-2 was examined in: (1) the RT4 bladder tumour cell line; (2) normal pig urothelium, and (3) human superficial bladder tumours. METHODS: In the RT4 cell line, uptake of FITC-AO, FITC-scrambled and FITC-sense oligonucleotides were quantified by flow cytometry at 4-hour intervals over 24 h. Uptake of FITC-AO was assessed in normal pig urothelium by flow cytometry after FITC-AO was infused for 1 h. Uptake of FITC AO was assessed in samples from 14 human superficial bladder tumours which were maintained in an ex vivo model. In samples from 6 tumours, uptake at 4 h was assessed using fluorescence microscopy. In samples from 8 separate tumours uptake every 4 h within the first 24-hour incubation period was assessed by flow cytometry. RESULTS: In the RT4 cell line the FITC-AO, FITC-scrambled and FITC-sense oligonucleotide uptake was similar. Disaggregated cells from the normal urothelium of the 3 pigs exhibited 33, 46 and 51% of cells staining positively for FITC-AO as determined by flow cytometry. All 6 tumour samples had detectable intracellular FITC-AO by fluorescence microscopy at 4 h. In the 8 tumours examined over the 24-hour incubation period, there was a range of percentages of positively staining cells. However, most tumours had a monotonic increase in intracellular fluorescence intensity that plateaued 16 h post-infusion. CONCLUSION: Antisense Bcl-2 oligonucleotides were readily taken up by superficial bladder cancer cells but the heterogeneous uptake in tumour samples needs to be considered when assessing the bioavailability of these drugs.

Extranodal marginal zone B-cell lymphoma genotyping by Alu-polymerase chain reaction.

DNA amplification by polymerase chain reaction (PCR) with primers designed on the widely distributed Alu sequences allows the production of specific inter-Alu DNA-fingerprints. Amplification of tumour and matched normal DNA can show differences due to genetic alterations within the tumour genome. We applied this approach to study low-grade extranodal marginal zone B-cell lymphoma (of MALT type). After digestion with restriction enzymes, DNA samples were separately amplified by PCR with three different Alu-primers. A comparison between the fingerprint pattern from lymphoma and normal samples was made. Inter-Alu bands differing between the two samples were excised from the gel, cloned and sequenced. Nine cases of low-grade MALT-lymphomas have been analysed, giving seventeen different bands between tumour and normal. DNA sequence analysis showed identities for three of them with sequences available at the GenBank. The methodology of Alu-PCR to detect DNA-based abnormalities, in addition or combination with RNA-based methods, is a powerful tool to identify candidate regions frequently altered in tumours. With the increased available genomic sequences through the Human Genome Project, there will be an increasing probability of picking up perfect homologies with these sequences using cloned differential Alu-PCR bands in BLAST searches through genome databases.

Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma.

PURPOSE: To evaluate the pharmacokinetics and toxicity of an antisense oligonucleotide targeting bcl-2 in patients with non-Hodgkin's lymphoma (NHL) and to determine efficacy using clinical and biologic end points. PATIENTS AND METHODS: Twenty-one patients with Bcl-2-positive relapsed NHL received a 14-day subcutaneous infusion of G3139, an 18-mer phosphorothioate oligonucleotide complementary to the first six codons of the bcl-2 open reading frame. Plasma pharmacokinetics were measured by anion exchange high-performance liquid chromatography. Response was assessed by computed tomography. Changes in Bcl-2 expression were measured by fluorescence-activated cell sorting of patients' tumor samples. RESULTS: Eight cohorts of patients received doses between 4. 6 and 195.8 mg/m(2)/d. No significant systemic toxicity was seen at doses up to 110.4 mg/m(2)/d. All patients displayed skin inflammation at the subcutaneous infusion site. Dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. The maximum-tolerated dose was 147.2 mg/m(2)/d. Plasma levels of G3139 equivalent to the efficacious plasma concentration in in vivo models were produced with doses above 36.8 mg/m(2)/d. Plasma levels associated with dose-limiting toxicity were greater than 4 microg/mL. By standard criteria, there was one complete response, 2 minor responses, nine cases of stable disease, and nine cases of progressive disease. Bcl-2 protein was reduced in seven of 16 assessable patients. This reduction occurred in tumor cells derived from lymph nodes in two patients and from peripheral blood or bone marrow mononuclear cell populations in the remaining five patients. CONCLUSION: Bcl-2 antisense therapy is feasible and shows potential for antitumor activity in NHL. Downregulation of Bcl-2 protein suggests a specific antisense mechanism.

Stochastic modeling of apoptosis tolerance distributions measured by multivariate flow analysis of human leukemia cells.

BACKGROUND: Cytofluorometric analysis allows single-cell resolution of all-or-none programmed cell death (apoptosis) responses and permits direct measurement of cumulative frequency distributions (CFDs) of apoptosis sensitivity from which the median apoptosis tolerance can be estimated. Robust estimation of susceptibility to apoptosis within neoplastic cell populations provides a means of either accurately determining pharmacologically induced changes in apoptosis sensitivity or comparing cell population responses to different apoptosis inducers. METHODS: Experimentally determined CFDs for VP-16 (etoposide)-induced apoptosis were measured by phosphotidylserine surface expression and mitochondrial membrane potential dissipation (DeltaPsi(m)) in BV173 leukemia cells. CFDs were modelled by a modified Hill equation using a four-parameter nonlinear regression from which median apoptosis tolerance (K) was estimated. RESULTS: Median apoptosis tolerance (K) was estimated from nonlinear regression analysis of CFDs for DeltaPsi(m) collapse and loss of membrane asymmetry. The error distribution of K determined from nonlinear regression analysis of 100 simulated CFDs was shown to exhibit an asymmetrical distribution. The asymmetrical likelihood intervals for K were computed iteratively, thereby providing a measure of experimental error. CONCLUSIONS: A distribution-based approach to apoptosis assay using multivariate flow analysis offers a powerful, quantitative technique for investigating the phenotypical basis of neoplastic cell responsiveness to apoptosis therapy, permitting separation of cell populations on the basis of apoptosis susceptibility.

Antisense therapy of hematologic malignancies.

Many tumor cells are inherently resistant to curative treatment due to an altered pattern of gene expression. It is an attractive and logical proposition to use this difference within the lymphoma cell to eradicate the malignant process. One such new therapeutic approach based on the "silencing" of genes involved in the prevention of apoptosis is Bcl-2 antisense oligonucleotide (AO) therapy. In the field of lymphoma, obvious targets included follicular lymphoma with the t(15;18) translocation, which results in deregulated expression of the Bcl-2 gene, chemoresistance, and subsequent protection against lymphoma cell death. Targeting the initiating codon of the Bcl-2 gene decreases both cell viability and Bcl-2 protein expression in lymphoma and leukemia cell lines that overexpress Bcl-2. Preclinical toxicity studies using a Bcl-2 AO G3139 (Genta, San Diego, CA) show good tolerance at a dose of 10 mg/kg, which is considerably higher than the dose required for good antilymphoma efficacy. In a phase I clinical study, G3139 was well tolerated with minimal toxicity in a dose escalation up to 147.2 mg/m2/d. Evidence of efficacy includes a responder with stage IVB follicular lymphoma who achieved complete clinical and radiologic response that has lasted more than 2 years. The main dose-limiting toxicity has been reversible thrombocytopenia related to the thioate backbone. Other antisense reagents are also in development to combat non-Hodgkin's lymphoma (NHL). These include oligonucleotides that target the messages of the Bcl-X(L) and protein kinase-Calpha (PKCalpha) genes. AOs may also have an application in tumors expressing mutant p53. AOs against MDM2 genes have shown the ability to restore wild-type p53 expression, suggesting that as oncogenic pathways are unraveled, normal cell growth and death patterns may be restored by molecular manipulation. Downregulation of antiapoptosis by AOs in the human setting has low toxicity and antilymphoma activity in cases in which conventional chemotherapy has failed. In the future, antisense therapy followed by chemotherapy may overcome chemoresistance to provide effective therapy for a range of malignancies.

Molecular genetics of extranodal marginal zone (MALT-type) B-cell lymphoma.

Mucosa-associated lymphoid tissue lymphoma is now classified as extranodal marginal zone B-cell lymphoma. We reviewed the current literature on the biological and genetic mechanisms that lead to the development and progression of this unusual lymphoma. Particular attention was given to the clinical and biological significance of the immunoglobulin genes rearrangement, that has been proposed and widely used both diagnostically and as a tool to monitor the response to antibiotic treatment.

Localization of Jacobsen syndrome breakpoints on a 40-Mb physical map of distal chromosome 11q.

Jacobsen syndrome is a haploinsufficiency disorder caused, most frequently by terminal deletion of part of the long arm of chromosome 11, with breakpoints in 11q23.3-11q24.2. Inheritance of an expanded p(CCG)n trinucleotide repeat at the folate-sensitive fragile site FRA11B has been implicated in the generation of the chromosome breakpoint in several Jacobsen syndrome patients. The majority of such breakpoints, however, map distal to this fragile site and are not linked with its expression. To characterize these distal breakpoints and ultimately to further investigate the mechanisms of chromosome breakage, a 40-Mb YAC contig covering the distal long arm of chromosome 11 was assembled. The utility of the YAC contig was demonstrated in three ways: (1) by rapidly mapping the breakpoints from two new Jacobsen syndrome patients using FISH; (2) by demonstrating conversion to high resolution PAC contigs after direct screening of PAC library filters with a YAC clone containing a Jacobsen syndrome breakpoint; and (3) by placing 23 Jacobsen syndrome breakpoints on the physical map. This analysis has suggested the existence of at least two new Jacobsen syndrome breakpoint cluster regions in distal chromosome 11.

Variability of polymerase chain reaction detection of the bcl-2-IgH translocation in an international multicentre study.

BACKGROUND: The capacity of the polymerase chain reaction (PCR) to detect very low numbers of cells bearing a t(14;18) translocation has led to its application in assessment of the results of treatment for follicular lymphoma, and suggestions that therapy might be guided by molecular studies. To test the reliability of PCR a collaborative study was undertaken to compare results from different laboratories in Europe and North America. METHODS: Twenty laboratories with records of publication in molecular diagnostics were sent blood from normal donors with varying numbers of t(14;18)-bearing cells added from a cell line with a translocation in the major breakpoint region (MBR) of the bcl-2 gene. Samples contained 1000, 100, 10, 1 or 0 cells per ml of whole blood and were sent blinded in duplicate. PCR methodology varied widely, with the total number of amplification cycles between 30 and 70, and 13 different primers used for the MBR region. Twelve laboratories used nested PCR and eight single round amplification. RESULTS: The sensitivity of nested and single round PCR was similar at 100 cells/ml but below this the nested method proved significantly more sensitive. The false positive rate was 28%, with 11 samples from 9 laboratories reported as positive when no t(14;18) cells were added. PCR product size and sequence analysis showed that false positives were due to contamination from cell-line DNA rather than background translocations in the donors. There was no significant difference in false positive rates between nested and single round techniques. CONCLUSION: The polymerase chain reaction to detect bcl-2-IgH rearrangements is presently carried out with widely disparate results. Further effort is required to bring forward a standard PCR protocol which can be re-tested in different laboratories to improve accuracy and reproducibility. The application of quantitative techniques such as real-time PCR may resolve many of the problems presently encountered.

Early postischemic dantrolene-induced amelioration of poly(ADP-ribose) polymerase-related bioenergetic failure in neonatal rat brain slices.

In the infant brain, ischemia-induced ionic and enzyme mechanisms may independently lead to cell death by energy depletion: resequestration of calcium mobilized from intracellular stores consumes ATP, and activated poly(ADP-ribose) polymerase (PARP) uses oxidized nicotinamide adenine dinucleotide to form polyADP-ribosyl nuclear proteins associated with DNA damage. Using 31P nuclear magnetic resonance spectroscopy, we have monitored intracellular pH and cellular energy metabolites in ex vivo neonatal rat cerebral cortex before, during, and after substrate and oxygen deprivation. In an insult that exhibited secondary energy failure and apoptosis we identified a relative 25% augmentation of high-energy phosphates at the end of recovery when the ryanodine-receptor antagonist, dantrolene, was introduced in the early (0- to 40-minute) but not late (40- to 120-minute) stage of recovery (P < 0.05). In contrast to the absence of a late dantrolene-sensitive effect, inhibition of PARP with 3-methoxybenzamide was as effective (P < 0.05) as early dantrolene, even when introduced after a 40-minute delay. The dantrolene and 3-methoxybenzamide effects on high-energy phosphates were not additive, rather the early dantrolene-sensitive effect nullified the potential 3-methoxybenzamide effect. Therefore, in this vascular-independent neonatal preparation, postischemic mobilization of calcium from intracellular stores is associated with PARP-related energy depletion. Inhibition of either of these processes confers improved postischemic bioenergetic recovery in the developing brain.