Modelling H3+ in planetary atmospheres: effects of vertical gradients on observed quantities.

Since its detection in the aurorae of Jupiter approximately 30 years ago, the H3+ ion has served as an invaluable probe of giant planet upper atmospheres. However, the vast majority of monitoring of planetary H3+ radiation has followed from observations that rely on deriving parameters from column-integrated paths through the emitting layer. Here, we investigate the effects of density and temperature gradients along such paths on the measured H3+ spectrum and its resulting interpretation. In a non-isothermal atmosphere, H3+ column densities retrieved from such observations are found to represent a lower limit, reduced by 20% or more from the true atmospheric value. Global simulations of Uranus' ionosphere reveal that measured H3+ temperature variations are often attributable to well-understood solar zenith angle effects rather than indications of real atmospheric variability. Finally, based on these insights, a preliminary method of deriving vertical temperature structure is demonstrated at Jupiter using model reproductions of electron density and H3+ measurements. The sheer diversity and uncertainty of conditions in planetary atmospheres prohibits this work from providing blanket quantitative correction factors; nonetheless, we illustrate a few simple ways in which the already formidable utility of H3+ observations in understanding planetary atmospheres can be enhanced. This article is part of a discussion meeting issue 'Advances in hydrogen molecular ions: H3+, H5+ and beyond'.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

Inhibition of BCL11B expression leads to apoptosis of malignant but not normal mature T cells.

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Krüppel-like zinc-finger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumor-suppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.

Disruption of the BCL11B gene through inv(14)(q11.2q32.31) results in the expression of BCL11B-TRDC fusion transcripts and is associated with the absence of wild-type BCL11B transcripts in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is associated with chromosomal aberrations characterized by juxtaposition of proto-oncogenes to T-cell receptor gene loci (TCR), resulting in the deregulated transcription of these proto-oncogenes. Here, we describe the molecular characterization of a novel chromosomal aberration, inv(14)(q11.2q32.31), in a T-ALL sample, involving the recently described BCL11B gene and the TCRD locus. The inversion joined the 5' part of BCL11B, including exons 1-3, to the TRDD3 gene segment of the TCRD locus, whereas the reciprocal breakpoint fused the TRDV1 gene segment to the fourth exon of BCL11B. The TRDV1-BCL11B joining region was 1344 bp long and contained fragments derived from 20q11.22, 3p21.33 and from 11p12, indicating the complex character of this aberration. A strong expression of in-frame transcripts with truncated BCL11B and TCRD constant region (TRDC) were observed, but in contrast to normal T cells and other T-ALL samples, no wild-type BCL11B transcripts were detected in the T-ALL sample. Screening of 37 other T-ALLs revealed one additional case with expression of the BCL11B-TRDC fusion transcript. As BCL11B appears to play a key role in T-cell differentiation, BCL11B disruption and disturbed expression may contribute to the development of T-cell malignancies in man.

Genetic characterization of the multidrug-resistant phenotype of VM-26-resistant human leukemic cells.

Our human T-cell leukemia line, CEM/VM-1, selected for resistance to VM-26 (teniposide), is cross-resistant to several drugs that interact with topoisomerase II, including VP-16 (etoposide), 4'-(9-acridinylamino)methanesulphon-m-anisidide, daunorubicin, and mitoxantrone. However, in contrast to cell lines exhibiting multidrug resistance (MDR) associated with overexpression of P-glycoprotein, this line is not cross-resistant to the Vinca alkaloids, is not impaired in drug accumulation, and does not overexpress the mdrl gene (Cancer Res., 47: 1297, 5455, 1987). More recently we found that nuclear extracts of these cells exhibit decreased topoisomerase II catalytic and cleavage activity, compared to the drug-sensitive line (Biochemistry, 1988). These results suggest that an alteration in topoisomerase II or a modulator of this enzyme may be responsible for this altered topoisomerase II-form of multidrug resistance (at-MDR). In the present work, we studied the somatic cell genetics of at-MDR. We produced hybrid cell lines by polyethylene glycol-mediated fusion of the CEM/VM-1 line with a hypoxanthine-guanine phosphoribosyl transferase-deficient, ouabain-resistant CEM line (CEM.AG1.OU1.5) that exhibits VM-26 sensitivity. Ten of the hybrid lines that grew in selective medium were randomly chosen for expansion and four were analyzed for both DNA content by flow cytometry and VM-26 sensitivity in a 72-h growth inhibition assay. The hybrid lines all contained approximately 2x DNA compared to unfused controls, indicating that the fusions were successful. The IC50 for VM-26 in 3 of the 4 lines was the same as that of the sensitive controls, ranging from 4.7 to 7.4 x 10(-8) M, and another was 76 x 10(-8) M. These data indicate that drug sensitivity was reconstituted by the hybridization procedure. By comparison, the VM-26 IC50 values in the CEM/VM-1 cells and CEM/VM-1 x CEM/VM-1 control "fusions" were 360 and 750 x 10(-8) M, respectively. To determine whether a topoisomerase II-mediated function was reconstituted in the hybrids, we measured drug-stimulated DNA cleavage ("cleavable complex formation"). Using 32P-labeled pBR322 DNA as substrate with nuclear extracts from drug sensitive cells, 100 microM VM-26 maximally stimulated DNA cleavage by approximately 11-fold compared to no-drug controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Decreased DNA topoisomerase II in daunorubicin-resistant Ehrlich ascites tumor cells.

We have previously shown that the multidrug-resistant EHR2/DNR+ cells, which overexpress P-glycoprotein, accumulate only about 20-30% of daunorubicin at steady state compared to the sensitive cells. These cells have been thought to be a "pure" P-glycoprotein cell line. We now report that the EHR2/DNR+ cells exhibit decreased DNA topoisomerase II catalytic activity. We also found that the amount of immunoreactive DNA topoisomerase II from these cells is about one-third that seen in the drug-sensitive cell line. In agreement with the decreased activity and amount of topoisomerase II, the number of DNA-protein complexes stabilized by teniposide (VM-26) was reduced by about 50% in nuclear extracts from EHR2/DNR+ cells. Furthermore, using an intact cell assay for DNA protein complexes, we found that the VM-26-stimulated complexes formed in the drug-resistant cells never reached the level seen in the drug-sensitive cells. Verapamil and Cremophor EL block P-glycoprotein-mediated efflux of "natural product" drugs and increase their accumulation in resistant cells. Coincubation of the EHR2/DNR+ cells with VM-26 and either of these modulators increased the number of complexes formed in the resistant cells. However, neither modulator increased the number of topoisomerase II-DNA complexes in the drug-resistant cells to the level seen in the EHR2 cells. We conclude that the resistance of EHR2/DNR+ cells is due in part to reduced amounts of DNA topoisomerase II. Furthermore, we note that a single cell line can express features of both P-glycoprotein-associated multidrug resistance and altered topoisomerase II-associated multidrug resistance.

LightCycler technology for the quantitation of bcr/abl fusion transcripts.

Quantifying bcr/abl fusion transcripts in chronic myelogenous leukemia is thought to serve as a powerful parameter for monitoring the kinetic nature of this clonal disease in vivo and in vitro. Recently, we demonstrated the technical advantages as well as the clinical relevance of quantitating bcr/abl fusion mRNA using the 5-nuclease assay and a real-time fluorescence reverse transcriptase-PCR (RT-PCR) detection system (ABI PRISM 7700 SDS). Meanwhile, another technique was introduced (LightCycler technology) that may be used for the same purpose. To investigate whether this method may be an appropriate alternative to the described procedure, we have established bcr/abl LightCycler RT-PCR for major and minor bcr/abl fusion transcripts. We found that, with only minor modifications, TaqMan RT-PCR and fluorescent probe design can be used to obtain comparable results in the LightCycler system. The developed method could quantitate as little as 10 bcr/abl copies per 100 ng cDNA and was as safe and reproducible as the previously described technique. Because reaction efficiency was identical within different bcr/abl major fusions, one single RT-PCR could be established that simultaneously detects b2a3, b2a2, b3a2, and b3a3 fusion RNA with equal specificity and sensitivity. Compared to results generated by the ABI PRISM 7700 SDS, absolute amounts of bcr/abl did not differ significantly, and there was a linear correlation between the respective values. We conclude that TaqMan chemistry can be used in the LightCycler and that both real-time fluorescence PCR detection systems equally fulfill the criteria for the safe and reliable quantitation of bcr/abl fusion RNA in clinical samples. This may be of help for further standardization of quantitative bcr/abl RT-PCR, which, again, is necessary for the comparison of results generated by different investigators.

Nuclear matrix antigens in azo dye-induced primary rat hepatomas.

Polyvalent antisera, monoclonal antibodies, and immunotransfer methodology have been used to identify and characterize a group of chromosomal protein antigens which appear during azo dye hepatocarcinogenesis. Experiments were designed to probe for the location and placement of antigens in chromatin according to solubility and possible DNA-binding properties. The majority of nuclear antigens were associated with high-speed DNA-containing pellets after ultracentrifugation of chromatin solubilized with denaturing buffers containing 6 M guanidine-HCl:2% sodium dodecyl sulfate, or 2 M NaCl:5 M urea. The addition of 2-mercaptoethanol or dithiothreitol to guanidine-HCl or sodium dodecyl sulfate solutions resulted in solubilization of nearly all antigens from the DNA pellets, suggesting the presence of complexes (protein:protein and/or DNA:protein) cross-linked with sulfhydryl linkages. Preparation of nuclear matrix from the primary hepatomas under several kinds of conditions indicated these antigens to be components of the residual nuclear matrix, envelope, and/or associated structures. Two-dimensional gel analysis showed most antigens to exist in a range of isoelectric forms, suggesting posttranslational modifications. Studies with monoclonal antibodies prepared to these proteins revealed extensive antigenic homology among the members comprising these fractions. Our results document antigenic differences in the nuclear matrix proteins of primary tumors and their normal tissue counterparts.

Characterization of monoclonal antibodies to novikoff hepatoma cytokeratin antigen p39.

Three stable monoclonal antibody-producing mouse hybridoma lines have been developed which produce high-titer, immunoglobulin M antibodies specific for the Novikoff ascites hepatoma (NAH) Mr 39,000 cytokeratin antigen (p39). Immunotransfer assays of cytoskeletal protein-enriched fractions indicated p39 to be present in a range of rat tissues, including colon, breast, lung, and uterus. Two-dimensional gel immunoblots confirmed that immunoreactivity in the latter tissues was for polypeptides with similar isoelectric points to those of NAH p39; however, reactivity in the colon contained a wide range of additional isomeric forms. Immunohistochemical localization studies with these antibodies revealed enrichment of p39 in the simple epithelia or ductular structures of organs containing this antigen. In all sections examined, glandular epithelia were observed to be only weakly immunoreactive. Additional immunoblot and immunocytochemical analyses with the monoclonal antibodies and polyspecific antisera to NAH cytokeratin suggest the human Mr 40,000 cytokeratin to be similar to but not identical to NAH p39.

Molecular quantification of response to therapy and remission status in TEL-AML1-positive childhood ALL by real-time reverse transcription polymerase chain reaction.

Although TEL-AML1 positivity [translocation t(12;21)(p13;q22)], detected in 20-25% of initial childhood acute lymphoblastic leukemia (ALL), has been associated with an excellent prognosis, its positive predictive value is insufficient for appropriate treatment stratification considering reported prevalence in relapsed ALL (3-28%). Molecular quantification of response to therapy by PCR-based methods has been shown to improve risk assessment. Here, we report on the sensitive quantification of leukemia-specific TEL-AML1 fusion transcript levels normalized to beta-actin expression (sensitivity threshholds, 10(-5)) by a novel real-time reverse transcription-PCR (RQ-RT-PCR) based on fluorescent TaqMan technique providing early and rapid evidence on the treatment efficacy of children with initial or relapsed TEL-AML1+ ALL enrolled in frontline or relapse trials of the Berlin-Frankfurt-Münster (BFM)-Study Group. In initial ALL, TEL-AML1/beta-actin decrease was > or =10(5)-fold in 50% of patients after induction therapy (day 33) and stayed TEL-AML1-negative throughout therapy, which suggested high sensitivity of leukemic cells to antineoplastic therapy. The remaining patients were still TEL-AML1+ before reintensification (ratios, 0.7 x 10(-2):10(-4)). In relapsed ALL, TEL-AML1/beta-actin decrease was generally less pronounced at corresponding time points, and conversion to TEL-AML1 negativity was observed in 40% of patients. Most notably, subsequent relapses occurred only among molecular poor responders, whereas all early responders remain in their second complete remission. In conclusion, real-time quantification of TEL-AML1/beta-actin kinetics distinguishes distinct molecular response groups, and provides indications capable of directing therapeutic interventions for patients with TEL-AML1+ ALL. Before considering modification of therapy, results should be interpreted cautiously taking into account the long duration of remission associated with TEL-AML1+ ALL.

Distinct expression patterns of the p53-homologue p73 in malignant and normal hematopoiesis assessed by a novel real-time reverse transcription-polymerase chain reaction assay and protein analysis.

The role of the recently identified first p53-homologue, p73, in neoplastic transformation is unknown. To elucidate p73 gene expression in hematopoiesis, we investigated samples from chronic myeloid leukemia (CML) and acute myeloid leukemia patients, leukemia cell lines, as well as mature and immature normal hematopoietic cells by real-time quantitative RT-PCR and Western blot analysis. We found a distinct p73 expression profile with highest p73 mRNA transcript levels in hematopoietic malignancies such as CML blast crisis and acute myelogenous leukemia versus CML chronic phase and normal controls. Mono- and biallelic p73 expression was found in both normal and malignant hematopoiesis. p73 protein was expressed at various levels in leukemia samples and cell lines but could not be detected in any normal controls tested. Our results point to a distinct yet undefined role of p73 in the pathogenesis of myeloid neoplasms.

A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing.

Recent improvements in high-throughput sequencing technologies underscore the pervasiveness of circular RNA (circRNA) expression in animal cells. CircRNAs are distinct from their linear counterparts because they lack the 5' caps and 3' tails that typically help determine the cellular fate of a transcript. However, due to the lack of free ends, circRNAs are impervious to exonucleases and thus can evade normal RNA turnover mechanisms. Most circRNAs are derived from protein-coding pre-mRNAs, via a mechanism called "back-splicing." Existing methods of circRNA expression thus typically involve genes that have been engineered to contain sequence elements that promote back-splicing. We recently uncovered an anciently conserved mechanism of RNA circularization in metazoans that involves splicing of tRNA introns. This splicing mechanism is completely independent from that of pre-mRNAs. In this chapter, we detail an orthogonal method that involves splicing of intron-containing tRNAs in order to produce circRNAs in vivo. We utilize fluorescence-based RNA reporters to characterize the expression, localization, and stability of these so-called tRNA intronic circular RNAs. Because tRNA biogenesis is essential for all cellular life, this method provides a means to express ultrastable, high-copy, circRNA effectors in a wide variety of metazoan cell types.

Demonstration of chimerism after allogeneic bone marrow transplantation by polymerase chain reaction of Y-chromosome-specific nucleotide sequences--characterization of a new technical approach.

Various techniques are applied to assess chimerism after allogeneic bone marrow transplantation. The polymerase chain reaction (PCR) of donor- and/or host-specific gene sequences provides a rapid and highly sensitive technique. We describe the characterization and application of PCR for the amplification of Y-chromosome-specific DNA in blood cells recovered from stored slides. Four different primer pair combinations were used. PCR can be rapidly performed on stained or unstained slide material with varying sensitivity--depending on the primer combination. The lowest limit of detection is one male cell in 500-1000 female cells. The technique was applied to follow the early post-transplant course of 15 male patients who received grafts from female donors and found a high incidence of mixed chimerism during the first three months after BMT and a striking fluctuation between positive and negative results in the follow-up of individual patients. We conclude that PCR for the detection of male-specific DNA sequences can be successfully performed with high sensitivity on material recovered from stored blood slides.

Ras point mutations occur in acute myeloid leukemia with illegitimate T-cell receptor delta gene rearrangement.

Mutations in the ras proto-oncogenes are the most frequently observed molecular alteration in acute myeloid leukemia (AML). Whether ras mutations occur as late or relatively early events in the multistep process of myeloid transformation, remains an open question. We previously described illegitimate T-cell receptor (TCR)-delta gene rearrangements in a subset of AML. These recombinations were detected in 9 out of 100 de novo AML cases. Southern blot analysis suggested the presence of these recombinations in the vast majority of AML cells and thus could be used as clonal markers. In order to more accurately define the role of ras proto-oncogene mutations in the multistep process of malignant transformation in myeloid leukemias, we performed single strand conformation polymorphism (SSCP) assays, slot blot and direct sequencing analysis on these nine cases with illegitimate TCR delta gene rearrangements. Ras proto-oncogene mutations were found in three of nine cases. Interestingly, SSCP, slot blot and sequencing suggested the presence of the respective mutations in most of the leukemic cells. Thus, ras mutations presumably occurred early in the process of transformation in these three cases.

Induction of differentiation in Friend-erythroleukemia cells by aclacinomycin A: early transient decrease in c-myc and c-myb mRNA levels.

Chemical inducers of the differentiation are known to cause an early transient decrease in c-myc and c-myb mRNA levels in Friend erythroleukemia cells preceding the down-regulation of c-myc and c-myb expression in the course of irreversible terminal differentiation. We therefore investigated the early effect of the potent differentiation-inducing anthracycline antitumor antibiotic, aclacinomycin A, on the c-myc and c-myb mRNA levels in the Friend cell line, F4-6, using Northern blot analysis. Aclacinomycin A induced a rapid decrease in the levels of c-myc and c-myb transcripts within 0.5-1 h and 2-3 h, respectively. The time course of decline in c-myc and c-myb expression was similar to that observed with dimethylsulfoxide or after transcription blockage brought about by a high concentration of actinomycin D. By 12 to 18 h after aclacinomycin A exposure, the c-myc and c-myb mRNA levels had returned to about pretreatment levels. When the cells were treated with adriamycin, an anthracycline that reduces cell proliferation in F4-6 cells without increasing differentiation, an early decrease in c-myc and c-myb expression was not observed. These results suggest that the transient decrease in c-myc and c-myb mRNA levels in F4-6 cells may be an early differentiation-related biochemical effect of aclacinomycin A.

Genetic alterations in the p53 gene in the blast crisis of chronic myelogenous leukemia: analysis by polymerase chain reaction based techniques.

Rearrangements of the c-abl protooncogene and the bcr-gene are found in > 90% of patients in chronic phase of chronic myelogenous leukemia (CML). The molecular events leading to blast crisis, however, have not been well characterized. Gross alterations of the p53 gene have been detected in 30% of patients with blast crisis. Since point mutations in the p53 gene appear to be important in the process of transformation in many epithelial tumors, we looked for these mutations in the critical regions of the p53 gene (exons 4, 5, 6, 7, and 8). We used the polymerase chain reaction (PCR), direct sequencing, differential PCR, and single strand conformation polymorphism (SSCP) analysis to detect mutations of the p53 gene in samples from 21 patients with CML blast crisis. Two of 21 patients exhibited an intragenic deletion or rearrangement in p53. In addition, these patients were homozygous for the mutant p53 allele. No mutations were found in the p53 gene of the remaining 19 patients. However, sequencing of the CML blast crisis cell line, K562, revealed an insertion of a C at base position 956 within the fifth exon, causing a frame shift mutation and an early translational stop at codon 148. We conclude that, in contrast to solid tumors, mutations in exons 4-8 of p53 are not frequently seen in primary samples from CML blast crisis. However, deletions and/or rearrangements within the p53 gene do occur and may contribute to the progression from chronic phase to blast crisis in a limited number of patients with CML.

Rearrangements of T-cell receptor delta, gamma and beta genes in acute myeloid leukemia coexpressing T-lymphoid features.

In order to investigate the role of T-cell receptor (TcR)-delta and TcR-gamma gene rearrangements and/or deletions in acute myeloid leukemia (AML) coexpressing T-cell-associated antigens (i.e. CD2 and/or CD4 and/or CD7), we examined blasts from a selected group of 56 AML cases (25 children, 31 adults) coexpressing either of these antigens without cytoplasmic CD3 expression. Forty-four typical AML cases (7 children, 37 adults) without T-cell associated antigens were further studied as controls. Germline configuration of the TcR-delta gene was observed in 91 out of the total of 100 AML cases investigated. Eight of nine cases with rearranged or deleted TcR-delta genes coexpressed T-cell-associated antigens. Blast cells of 7/9 cases were classified as FAB M1, two as FAB M2. In six of these cases TcR-gamma gene rearrangements were also detected. TcR-delta alterations were predominantly found in children whose blasts coexpressed T-lymphoid associated antigens (6/25, 24%), but were rarely detected in adult AML with or without coexpression of T-cell antigens (2/31 and 0/37, respectively).

Quality assurance in RT-PCR-based BCR/ABL diagnostics--results of an interlaboratory test and a standardization approach.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Elevated p21 mRNA level in skeletal muscle of DMD patients and mdx mice indicates either an exhausted satellite cell pool or a higher p21 expression in dystrophin-deficient cells per se.

Abnormalities in proliferation and differentiation of the dystrophin-deficient muscle are a controversial aspect of the pathogenesis of Duchenne muscular dystrophy (DMD). Analyses of molecules involved in cell cycle modulation do not exist in this context. Cells withdrawn from the cell cycle permanently express p21. The fact that p2 1, in contrast to other cell cycle proteins, is not diminished when myotubes are reexposed to growth media, allocates this cyclin-dependent kinase inhibitor a special function. Here we report for the first time statistically increased p21 mRNA levels in dystrophin-deficient muscle tissue. Only 42% of conventional RT-PCRs from six muscle samples of human controls yielded positive results but almost all skeletal muscle biopsy samples (87%) from DMD patients (n=5). For p21 mRNA quantification in murine muscle samples we were able to use the exact real-time TaqMan PCR method due to generally higher p21 mRNA levels than in human muscles. In addition, contamination with fibroblasts can be excluded for the murine samples because they do not demonstrate fibrosis at the age of 350 days but start to lose their regenerative capacity. In accord with the results in humans, we observed p21 mRNA levels in mdx mice that were approx. four times as high as those in control mice. Elevated p21 mRNA level may indicate a shift in cell composition towards differentiated p21 expressing cells as a result of an exhausted pool of undifferentiated, non-p21-expressing satellite cells due to previous cycles of de- and regeneration. Alternatively, dystrophin-deficient cells per se may express higher p21 levels for unknown reasons. Although we cannot distinguish between these possibilities, the eventual transfec tion of a patient's own satellite cells with p21 antisense oligonucleotides may enable the dystrophic process to be influenced.

Selection of B-cell chronic lymphocytic leukemia cell variants by therapy with anti-CD20 monoclonal antibody rituximab.

OBJECTIVE: Anti-CD20 chimeric monoclonal antibody rituximab (Mabthera; IDEC-C2B8) is currently tested in several clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). In the present study, we investigated whether rituximab therapy may select for CD20(-) subclones. MATERIALS AND METHODS: Leukemic B-CLL cells were isolated from patients with B-CLL and sensitivity to rituximab-induced cell death was examined. Levels of CD20 protein and mRNA were determined using flow cytometry and real-time PCR, respectively. Clonality analyses of leukemic cells throughout rituximab therapy were performed by GeneScan analysis of patient clone specific rearrangements of the complementarity determining region III of the heavy chain immunoglobulin. RESULTS: Cytotoxicity of rituximab in vitro did not depend on the protein levels of CD20. During therapy with rituximab CD20(+) B-CLL cells were depleted and CD20(-) leukemic cells emerged. After treatment, the initial CD20(+) B-CLL cell clone reexpanded. CD20(-) B-CLL cells retained their capacity to synthesize the CD20 molecule. CONCLUSIONS: These data support the concept that in B-CLL rituximab treatment may not lead to the emergence of CD20(-) leukemic variants. Our findings support clinical studies investigating the benefit of prolonged period of rituximab therapy in B-CLL disease.