In vitro and in vivo evaluation of the haematopoietic potential of skeletal muscle in a non-human primate model.

This study was aimed at evaluating the in vitro and in vivo haematopoietic potential in macaque skeletal muscle cells. Biopsy samples showed the presence of CD34(+) (7.6%), CD90(+) (8.4%), CD117(+), CD31(+), side population (SP) cells (7-10%) and a low number of CD45(+) cells. In clonogenic and long-term culture-initiating cell assays, no haematopoietic potential could be detected in either total mononuclear cells or SP cells. Regarding in vivo studies, two animals were transplanted with unfractionated fresh muscle cells after lethal irradiation. Both animals died early after transplant without any evidence of haematopoietic reconstitution. In two other monkeys, harvested muscle cells were frozen and secondarily marked using a green fluorescent protein (GFP)-lentiviral vector. After sublethal irradiation, both animals were transplanted with GFP-expressing muscle cells followed by a bone marrow rescue. Both animals had haematopoietic reconstitution at days 22 and 25, but no GFP-expressing haematopoietic cells could be detected by flow cytometry, either in the blood or in clonogenic cells from marrow aspirates. Using PCR assays, GFP(+) cells were detected in a single marrow sample of one animal at 41 days after transplantation. These results strongly suggest that as opposed to murine muscle, the non-human primate skeletal muscle does not harbour cells with a straightforward haematopoietic potential.

Transient suppression of clonal hemopoiesis associated with pregnancy in a patient with a myeloproliferative disorder.

We have used restriction fragment length polymorphism analysis to study the clonal involvement of the blood cells in a woman with myeloproliferative disease, whose initially high platelet count (940,000/microliter) spontaneously decreased during a normal pregnancy but then returned rapidly to the same high level after delivery of her child. Analysis of her erythroid progenitors showed the presence of erythropoietin-independent progenitors before, during, and after her pregnancy, consistent with a diagnosis of myeloproliferative disease, and persistence of the abnormal clone throughout the period of study. Analysis of DNA from her blood granulocytes showed these to be polyclonal at mid-pregnancy, when her platelet count had decreased to normal values, in comparison to the monoclonal pattern exhibited by her blood granulocytes 3 mo postpartum, when her platelet count was again elevated. These results demonstrate a partial conversion to normal, polyclonal hemopoiesis during her pregnancy and suggest a previously unanticipated differential sensitivity of normal and neoplastic hemopoietic cells to physiological changes associated with this state.

Familial bone marrow monosomy 7. Evidence that the predisposing locus is not on the long arm of chromosome 7.

Loss of expression of a tumor-suppressing gene is an attractive model to explain the cytogenetic and epidemiologic features of cases of myelodysplasia and acute myelogenous leukemia (AML) associated with bone marrow monosomy 7 or partial deletion of the long arm (7q-). We used probes from within the breakpoint region on 7q-chromosomes (7q22-34) that detect restriction fragment length polymorphisms (RFLPs) to investigate three families in which two siblings developed myelodysplasia with monosomy 7. In the first family, probes from the proximal part of this region identified DNA derived from the same maternal chromosome in both leukemias. The RFLPs in these siblings diverged at the more distal J3.11 marker due to a mitotic recombination in one patient, a result that suggested a critical region on 7q proximal to probe J3.11. Detailed RFLP mapping of the implicated region was then performed in two additional unrelated pairs of affected siblings. In these families, DNA derived from different parental chromosome 7s was retained in the leukemic bone marrows of the siblings. We conclude that the familial predisposition to myelodysplasia is not located within a consistently deleted segment on the long arm of chromosome 7. These data provide evidence implicating multiple genetic events in the pathogenesis of myelodysplasia seen in association with bone marrow monosomy 7 or 7q-.

Generation of an induced pluripotent stem cell line from a patient with hereditary multiple endocrine neoplasia 2B (MEN2B) syndrome with "highest risk" RET mutation.

Multiple Endocrine Neoplasia Type 2B (MEN2B) is a cancer-predisposing syndrome that affects patients with germline RET mutations. The clinical spectrum of the syndrome includes medullary thyroid carcinoma (MTC) and pheochromocytoma. Currently, there is no satisfactory model recapitulating all the features of the disease especially at the level of stem cells. We generated induced pluripotent stem cells (iPSCs) from a patient with RET mutation at codon 918 who developed pheochromocytoma and MTC. These iPSC had normal karyotype, harboured the RETM918T mutation and expressed pluripotency hallmarks. A comprehensive pathological assessment of teratoma was performed after injection in immunodeficient mice.

Generation of an induced pluripotent stem cell line from a patient with chronic myeloid leukemia (CML) resistant to targeted therapies.

Chronic myeloid leukemia (CML) is a clonal malignancy initiated by the occurrence of a t (9;22) translocation, generating Ph1 chromosome and BCR-ABL oncogene in a primitive hematopoietic stem cell (HSC). The resistance of HSC to targeted therapies using tyrosine kinase inhibitors remains a major obstacle towards the cure. We have generated an iPSC line from a patient with CML using leukemic CD34+ cells cryopreserved at diagnosis. Ph1+ CML cells were reprogrammed by non-integrative viral transduction. These iPSCs harboured Ph1 chromosome and expressed pluripotency hallmarks as well as BCR-ABL. Teratoma assays revealed normal differentiation after injection in immunodeficient mice.

Generation of an induced pluripotent stem cell line from a patient with hereditary multiple endocrine neoplasia 2A (MEN2A) syndrome with RET mutation.

Multiple Endocrine Neoplasia Type 2A (MEN2A) is a cancer-predisposing syndrome that affects patients with germline RET mutations. The clinical spectrum of the syndrome includes medullary thyroid carcinoma (MTC), pheochromocytoma, hyperparathyroidism and cutaneous lichen amyloidosis (CLA) and/or Hirschsprung disease in some variants. Currently, there is no satisfactory animal model recapitulating all the features of the disease especially at the level of stem cells. We generated induced pluripotent stem cells (iPSCs) from a patient with RET mutation at codon 634 who developed pheochromocytoma and MTC. RETC634Y-mutated cells were reprogrammed by non-integrative viral transduction. These iPSCs had normal karyotype, harboured the RETC634Y mutation and expressed pluripotency hallmarks as well as RET. A comprehensive pathological assessment of teratoma was performed after injection in immunodeficient mice.

The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways.

Inappropriate activation of Abl family kinases plays a crucial role in different human leukaemias. In addition to the well known oncoproteins p190Bcr-Abl and p210Bcr-Abl, Tel-Abl, a novel fusion protein resulting from a different chromosomal translocation, has recently been described. In this study, the kinase specificities of the Bcr-Abl and Tel-Abl proteins were compared to the physiological Abl family kinases c-Abl and Arg (abl related gene). Using short peptides which correspond to the target epitopes in known substrate proteins of Abl family kinases, we found a higher catalytic promiscuity of Bcr-Abl and Tel-Abl. Similar to Bcr-Abl, Tel-Abl was found in complexes with the adapter protein CRKL. In addition, c-Crk II and CRKL are tyrosine phosphorylated and complexed with numerous other tyrosine phosphorylated proteins in Tel-Abl expressing Ba/F3 cells. GTPase analysis with a Ras-GTP-specific precipitation assay showed constitutive elevation of GTP-loaded Ras in cells expressing the leukaemic Abl proteins. The mitogenic MAPK/Erk kinases as well as Akt/PKB, a kinase implicated to negatively regulate apoptosis, were also constitutively activated by both Bcr-Abl and Tel-Abl. The results indicate that the leukaemic Abl-fusion proteins have catalytic specificities different from the normal kinases c-Abl and Arg and that Tel-Abl is capable to activate at least some pathways which are also upregulated by Bcr-Abl.

BCR-ABL and constitutively active erythropoietin receptor (cEpoR) activate distinct mechanisms for growth factor-independence and inhibition of apoptosis in Ba/F3 cell line.

The interleukin-3 dependent murine Ba/F3 cell line has been widely used as an experimental model of cell transformation by BCR-ABL oncogenes as assessed by induction of growth-factor-independence and inhibition of apoptosis in vitro. The signaling pathways used by BCR-ABL oncogenes to exert these effects are unknown. To gain insights into this phenomenon, we have introduced the p190- and p210-encoding BCR-ABL oncogenes as well as the constitutively activated oncogenic murine erythropoietin receptor (cEpoR) into Ba/F3 and compared the behavior of individual clones in response to apoptotic stimuli. Both p210 and p190 BCR-ABL vectors induced IL-3-independent growth and the same result was obtained with the cEpo-R vector. Individual clones of Ba/F3 cells expressing BCR-ABL exhibited significant resistance to apoptosis induced by either etoposide, serum deprivation or growth-factor withdrawal. In contrast, Ba/F3 cells expressing the constitutively active cEpoR behaved like parental Ba/F3 cells undergoing apoptosis when similarly treated with etoposide or upon serum deprivation. Bc12 and Bax levels were similar in all BCR-ABL and cEpoR-transfected clones. However, in band-shift assays, nuclear extracts from growth-factor-independent Ba/F3 clones expressing cEpoR had no detectable STAT activity as opposed to the constitutive STAT activation detected in all Ba/F3 clones expressing p210 or p190 BCR-ABL. Our results indicate that although both constitutively activated cEpoR and BCR-ABL oncogenes induce growth-factor independence in Ba/F3 cells, only BCR-ABL is able to protect cells from etoposide and serum-deprivation-induced apoptosis and induce a strong constitutive activation of STAT factors, suggesting a role for these molecules in the anti-apoptotic activity of BCR-ABL.

Mutator phenotype of BCR--ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase beta.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome resulting from the translocation t(9-22) producing the chimeric 190 and 210 kDa BCR-ABL fusion proteins. Evolution of the CML to the more agressive acute myelogenous leukemia (AML) is accompanied by increased cellular proliferation and genomic instability at the cytogenetic level. We hypothezised that genomic instability at the nucleotide level and spontaneous error in DNA replication may also contribute to the evolution of CML to AML. Murine Ba/F3 cell line was transfected with the p190 and p210-encoding BCR-ABL oncogenes, and spontaneous mutation frequency at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci were measured. A significant 3-5-fold increase in mutation frequency for the transfected cells relative to the untransfected control cells was found. Furthermore, we observed that BCR-ABL transfection induced an overexpression of DNA polymerase beta, the most inaccurate of the mammalian DNA polymerases, as well as an increase in its activity, suggesting that inaccuracy of DNA replication may account for the observed mutator phenotype. These data suggest that the Philadelphia abnormality confers a mutator phenotype and may have implications for the potential role of DNA polymerase beta in this process.

Multiple molecular mechanisms contribute to radiation sensitivity in mantle cell lymphoma.

Mantle cell lymphomas (MCL) are characterized by their aggressive behavior and poor response to chemotherapy regimens. We report here evidence of increased in vitro radiation sensitivity in two cell lines that we have generated from two MCL patients (UPN1 and UPN2). However, despite their increased radiation sensitivity, UPN2 cells were totally resistant to apoptotic cell death, whereas UPN1 cells underwent massive apoptosis 6 h after irradiation. The frequency of induced chromosomal abnormalities was higher in UPN1 as compared to UPN2. Distinct mechanisms have been found to contribute to this phenotype: a major telomere shortening (UPN1 and UPN2), deletion of one ATM allele and a point mutation in the remaining allele in UPN2, mutation of p53 gene (UPN1 and UPN2) with absence of functional p53 as revealed by functional yeast assays. After irradiation, Ku70 levels in UPN1 increased and decreased in UPN2, whereas in the same conditions, DNA-PKcs protein levels decreased in UPN1 and remained unchanged in UPN2. Thus, irradiation-induced apoptotic cell death can occur despite the nonfunctional status of p53 (UPN1), suggesting activation of a unique pathway in MCL cells for the induction of this event. Overall, our study demonstrates that MCL cells show increased radiation sensitivity, which can be the result of distinct molecular events. These findings could clinically be exploited to increase the dismal response rates of MCL patients to the current chemotherapy regimens.

Quantitative non-radioactive clonality analysis of human leukemic cells and progenitors using the human androgen receptor (AR) gene.

Clonal analysis of FACS-purified primitive hematopoietic stem cells and of their progeny as assessed by the progenitors obtained from long-term cultures requires PCR-based approaches, mainly because of the low number of cells available. We have developed a non-radioactive androgen receptor (AR) assay which allows a simple and quantitative evaluation of the clonality of hematopoietic cells and progenitors. In this approach 5' AR primer is labelled by fluorescein and the amplified product is run on a sequencing gel which allows evaluation of the intensity of the fluorescent peaks generated. A computer software then analyzes the reduction of the intensity of the peaks on HpaII-digested samples. In order to determine the feasibility of the technique, we analyzed the clonality of leukemic cells from a patient with an acute-phase CMML which showed a typical clonal pattern of her leukemic DNA sample (WBC = 300 x 10(9)/I) using phosphoglycerate kinase (PGK) analysis. The same sample was then analyzed with either radioactive- or fluorescein-labelled AR primers, showing a typical clonal pattern (complete disappearance of one allele after HpaII digestion). A short-term clonogenic assay was then set up on methylcellulose and clonogenic progenitors were individually analyzed. All 24 colonies tested showed a typical clonal pattern with the disappearance of the same allele on each sample after HpaII digestion, indicating that they all derived from the same leukemic stem cell. Using this approach we then analyzed 94 patients with several hematologic malignancies and quantification of their fluorescent peaks. Fifty-four percent of the patients were clearly heterozygous (ie, a difference of > or = 2 CAG repeats was present between the two copies of the gene) and could be analyzed in an automatic sequencer using the fluorescent primers. Bone marrow mononuclear cells from all patients with acute myeloid leukemia (AML) showed a clonal or oligoclonal pattern at diagnosis whereas a polyclonal pattern was seen when remission was obtained. Similarly, out of 21 patients with a diagnosis of myelodysplastic syndrome (MDS), a clonal pattern was demonstrated in 10 whereas an oligoclonal or non-clonal pattern was shown in 11. These results show that this non-radioactive and safe technology can now be used on a large scale to evaluate the clonality of highly purified hematopoietic stem cells and their progenitors in hematopoietic malignancies and this might allow new insights into the targets of clonal amplification.

Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7.

There is currently no satisfactory model allowing analysis of dose-effect relationships of BCR-ABL proteins in human hematopoietic cells. To study comparatively the proliferative, differentiative and anti-apoptotic actions of different levels of BCR-ABL proteins in the context of the same cellular background, we have introduced the BCR-ABL gene into the GM-CSF-dependent pluripotent human cell line UT-7. Individual clones expressing BCR-ABL were analyzed by Western blots. After normalization to equivalent levels of endogenous ABL protein, 14 clones always grown in GM-CSF were found to express low but variable levels of BCR-ABL whereas two clones selected in the absence of GM-CSF expressed very high levels of BCR-ABL. All low-level BCR-ABL expressing clones exhibited a behavior similar to that of the GM-CSF-dependent parental cells as they ceased to proliferate upon growth factor deprivation and showed a strong proliferative response upon GM-CSF addition. One out of 14 clones showed progressive GM-CSF independence during culture over several weeks and was found to have a significant increase of BCR-ABL expression at that time. The resistance of this clone (E8-2) to different apoptotic stimuli was found to be increased as compared to its low BCR-ABL-expressing counterpart (E8-1) and similar to that observed in clones with very high levels of BCR-ABL (UT-7/9 and UT-7/11) which were totally resistant to apoptotic stimuli. When injected into nude mice, parental UT-7 cells and clones with low-level of BCR-ABL were not tumorigenic over 10 weeks of observation whereas UT-7 clones with high levels of BCR-ABL (UT-7/9, UT-7/11 and UT-7/E8-2) induced aggressive tumors in 2-4 weeks with a significant correlation between the amount of BCR-ABL protein and the rate of tumor growth. In conclusion, the establishment of an in vitro and in vivo CML model using UT-7 cells suggests for the first time in human cells, that the fully transformed phenotype induced by BCR-ABL requires high levels of BCR-ABL expression. These findings suggest that variable levels of BCR-ABL in primary patient cells could also be responsible for the different phenotypic features seen in chronic and acute phases of CML, such as the differentiation ability induced by growth factors.

Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette.

The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2-3 weeks. A single dose of doxycycline added to the medium (1 microg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60-90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 x 10(6) Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 microg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin- bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.

Efficient ex vivo expansion of NOD/SCID-repopulating cells with lympho-myeloid potential in hematopoietic grafts of children with solid tumors.

INTRODUCTION: The ex vivo expansion of hematopoietic grafts could be an important therapeutic tool for accelerating hematopoietic recovery after administration of high-dose chemotherapy regimens. The fate of the long-term repopulating cells during the ex vivo manipulation of grafts is a critical issue and will ultimately define the clinical applicability of this technology to hematopoietic transplantation. MATERIALS AND METHODS: To study the effects of a clinically applicable ex vivo expansion protocol in the proliferative potential of the most primitive human hematopoietic cells, both LTC-IC and NOD/SCID-RC assays were used to determine LTC-IC and NOD/SCID-RC contents of hematopoietic grafts, both before and after expansion (SCF, IL-3, PEG-MGDF Flt3-L and 5% AB serum), in four children with non-hematological malignancies. RESULTS: The mean percentage of CD34+ cells after expansion was 16%. The numbers of nucleated cells increased 20-fold with a mean three-fold increase in the numbers of CD34+ cells during the expansion period. The CFC content of the samples showed a mean 11-fold increase (range: 5-17) after ex vivo expansion. The primitive hematopoietic stem cell content of the expanded cell fraction evaluated by LTC-IC assays was found to be increased in two patients out of three, with maintenance of the LTC-IC frequency in the third patient. The NOD/SCID-RC potential, evaluated in five experiments from four patients using 109 mice injected 5-6 weeks earlier with human hematopoietic cells, increased from a mean percentage of 36% (range: 7-75%) before expansion, to a mean percentage of 70% (range: 37-100%) after expansion (P < 0.00001). The frequency of NOD/SCID-RC calculated with pooled data from all patients was 1/80,000 at day 0 and 1/40,000 after seven days of culture. The full phenotypic analysis of human hematopoietic cells obtained in NOD/SCID mice injected with expanded cells showed the presence of significant numbers of CD34+, CD19+ and CD15+ cells, suggesting the persistent lympho-myeloid potential of the expanded hematopoietic cells. CONCLUSION: Our results suggest that efficient expansion of NOD/SCID-RC with lympho-myeloid potential can be achieved not only in cord blood or normal marrow as previously reported, but also in hematopoietic grafts obtained from children exposed to high-dose chemotherapy.

Ex vivo expansion of megakaryocyte precursor cells in autologous stem cell transplantation for relapsed malignant lymphoma.

To evaluate the impact of ex vivo expanded megakaryocyte (MK) progenitors on high-dose chemotherapy-induced thrombocytopenia, we conducted a phase II study in 10 patients with relapsed lymphoma. Two fractions of peripheral blood progenitor cells (PBPC) were cryopreserved, one with enough cells for at least 2 x 10(6) CD34+ cells/kg and a second obtained after CD34+ selection. Ten days before autologous stem cell transplantation, the CD34+ fraction was cultured with MGDF+SCF for 10 days. After BEAM (BCNU, cyclophosphamide, cytarabine, and melphalan) chemotherapy, patients were reinfused with standard PBPC and ex vivo expanded cells. No toxicity was observed after reinfusion. The mean fold expansion was 9.27 for nucleated cells, 2 for CD34+ cells, 676 for CD41+ cells, and 627 for CD61+ cells. The median date of platelet transfusion independence was day 8 (range: 7-12). All patients received at least one platelet transfusion. In conclusion, ex vivo expansion of MK progenitors was feasible and safe, but this procedure did not prevent BEAM-induced thrombocytopenia. Future studies will determine if expansion of higher numbers of CD34+ cells towards the MK-differentiation pathway will translate into a functional effect in terms of shortening of BEAM-induced thrombocytopenia.

Molecular analysis of clonality in Kaposi's sarcoma.

BACKGROUND: Kaposi's sarcoma is considered to be an angioproliferative disease associated with a novel herpesvirus (KSHV/HHV8), but the precise pathophysiology of the lesion remains unclear. The study of clonality in Kaposi's sarcoma using X linked DNA polymorphism has been difficult so far, because of a very strong prevalence of the disease in males. AIMS: To study the clonality of Kaposi's sarcoma lesions. METHODS: An assay based on a methyl sensitive restriction digest followed by polymerase chain reaction (PCR) amplification of the highly polymorphic human androgen receptor (HUMARA) gene was used. Tissues from Kaposi's sarcoma lesions and control tissues from the same patients were obtained from seven females, four with classic Kaposi's sarcoma and three with AIDS associated Kaposi's sarcoma. A cutaneous angiosarcoma was also analysed, for comparative purposes, and showed evidence of clonality after HpaII digestion. RESULTS: All patients were heterozygous for the HUMARA polymorphism and informative for analysis. In all patients, including four with a nodular form of Kaposi's sarcoma and more than 70% spindle cells in the lesion, a polyclonal pattern of inactivation could be demonstrated. CONCLUSIONS: The Kaposi's sarcoma lesion is first of all a polyclonal cell proliferation.

Blastoid mantle cell lymphoma: evidence for nonrandom cytogenetic abnormalities additional to t(11;14) and generation of a mouse model.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32), which is associated with cyclin D1 hyperexpression and a poor prognosis. MCL cases have been shown to progress to a more aggressive disease but the molecular events responsible of this phenomenon have not been determined. We have established two cell lines from the pleural effusions of two patients with MCL that we have used for further cytogenetic characterization to better define the incidence and nature of secondary chromosome abnormalities using multicolor fluorescence in situ hybridization, whole chromosome paint, and specific probes. Both cell lines grew independently without growth factors. Using CCND1/IGH-specific probes, patient UPN1 was found to have a masked t(11;14). Numerous and complex chromosomal abnormalities were found in both cell lines affecting chromosomes 2, 8, 13, 18, 22, X, and Y. These abnormalities included 8p losses, suggesting the presence of an anti-oncogene in this region, rearrangements of 8q24, MYC gene, and translocations involving 8, X, and Y chromosomes, which might be significant in the pathogenesis of MCL progression. The use of the cell lines (UPN1) allowed us to generate a mouse model of human MCL, mimicking a disseminated lymphoma and leading to the death of the animals in 4 weeks. This blastoid MCL model could be of major interest to determine molecular events involved in MCL progression, allowing isolation of involved genes and their functional characterization, and to study the effects of new chemotherapy regimens in mouse models.

BCR-ABL fails to inhibit apoptosis in U937 myelomonocytic cells expressing a carboxyl-terminal truncated STAT5.

Recent experimental data suggest that one of the major effects of BCR-ABL gene expression in hematopoietic cells is the inhibition of apoptosis. Although the exact mechanisms of this phenomenon are not clear, it is thought to be related to the fact that BCR-ABL induces several signalling pathways also activated by growth factors. In order to determine the anti-apoptotic role of BCR-ABL in a hematopoietic cell line and to by-pass the influence of cytokine-dependence, BCR-ABL gene was expressed in the autonomously growing myelomonocytic U937 cell line using retroviral vectors. There was no resistance to apoptosis induced by either serum deprivation or different doses of etoposide in any U937 clones expressing BCR-ABL protein. In addition to serum deprivation and etoposide, BCR-ABL-expressing clones were not protected from apoptosis induced by TNF, ceramide-C2 and FAS-cross-linking. BCL2 expression was absent in U937 cells and BAX levels were identical between Neo and BCR-ABL clones. To further investigate the mechanisms of this phenomenon, band-shift assays were performed to detect activation of STAT molecules. No constitutive activation of STATs was detected in either NeoR or BCR-ABL-U937 cells, although both IFN-gamma and GM-CSF activated STAT1 and STAT5, respectively, with similar kinetics in both NeoR and BCR-ABL-U937 cells. In addition, the GM-CSF-induced-STAT5 activation was found to be weakened in all clones expressing BCR-ABL. In both control NeoR and BCR-ABL-transfected clones, band-shift assays revealed the presence of an abnormal truncated STAT5 recognized only by an anti-N-terminal but not by an anti-C-Terminal STAT5 antibody. These findings suggest a possible link between the absence of anti-apoptotic potential of BCR-ABL and abnormalities of the STAT5 pathway, including, absence of constitutive activation of STAT5, inhibition of GM-CSF-induced STAT5 activation and expression of a carboxyl-terminal-truncated STAT5.

Hematologic and immunologic changes in hairy cell leukemia patients treated with alpha interferon.

We prospectively evaluated hematologic and immunologic changes in fourteen hairy cell leukemia patients treated with alpha interferon. Prompt disappearance of circulating hairy cells, and recovery of peripheral blood counts was observed in all patients whereas only four patients had complete disappearance of hairy cell infiltrates from their bone marrow. Immunologic changes included a statistically significant increase of NK cell activity in the whole group and a slight decrease in circulating T cells with a strong trend towards an increase in the T4/T8 lymphocyte ratio (in eight of fourteen patients). Serial hemopoietic progenitor cell culture studies from peripheral blood (thirteen patients) and bone marrow (two patients) showed no correlation between hematologic recovery data and cell culture data.