CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome.

BACKGROUND: Russell Silver syndrome (RSS) leads to prenatal and postnatal growth retardation. About 55% of RSS patients present a loss-of-methylation of the paternal ICR1 domain on chromosome 11p15. CDKN1C is a cell proliferation inhibitor encoded by an imprinted gene in the 11p15 ICR2 domain. CDKN1C mutations lead to Beckwith Wiedemann syndrome (BWS, overgrowth syndrome) and in IMAGe syndrome which associates growth retardation and adrenal insufficiency. We searched for CDKN1C mutations in a cohort of clinically diagnosed RSS patients with no molecular anomaly. METHOD: The coding sequence and intron-exon boundaries of CDKN1C were analysed in 97 RSS patients. The impact of CDKN1C variants on the cell cycle in vitro were determined by flow cytometry. Stability of CDKN1C was studied by western immunoblotting after inhibition of translation with cycloheximide. RESULTS: We identified the novel c.836G>[G;T] (p.Arg279Leu) mutation in a familial case of intrauterine growth retardation (IUGR) with RSS phenotype and no evidence of IMAGe. All the RSS patients inherited this mutation from their mothers (consistent with monoallelic expression from the maternal allele of the gene). A mutation of this amino acid (p.Arg279Pro) has been reported in cases of IMAGe. Functional analysis showed that Arg279Leu (RSS) did not affect the cell cycle, whereas the Arg279Pro mutation (IMAGe) led to a gain of function. Arg279Leu (RSS) led to an increased stability which could explain an increased activity of CDKN1C. CONCLUSIONS: CDKN1C mutations cause dominant maternally transmitted RSS, completing the molecular mirror with BWS. CDKN1C should be investigated in cases with family history of RSS.

Evaluation of the effect of autologous mesenchymal stem cell injection in a large-animal model of bilateral kidney ischaemia reperfusion injury.

OBJECTIVES: Adult mesenchymal stem cells (MSC) have been proven to be of benefit to the kidney in different experimental models of renal injuries. All studies have been performed in valuable rodent models, but the relevance of these results to large mammals and ultimately, to humans remains unknown. Therefore, the aim of this study was to investigate the effect of MSC transplantation in an alternative ovine large-animal model of bilateral kidney ischaemia reperfusion injury. MATERIAL AND METHODS: Sheep were divided into three groups: one sham-operated group and two groups submitted to renal bilateral ischaemia for 60 min. Animals with ischaemia reperfusion injury were treated with injection of autologous MSCs or with vehicle medium. RESULTS: The model sheep presented with renal histological manefestations that closely resembled lesions seen in patients. Transplanted MSCs were found in glomeruli but not in tubules and did not express glomerular cell markers (podocin, von Willebrand factor), but functional evaluation showed no beneficial effect of MSC infusion. Morphological and molecular analyses corroborated the functional results. MSCs did not repair kidney parenchyma and failed to modulate cell death and proliferation or cytokine release (tumour necrosis factor-alpha, vascular endothelial growth factor alpha (VEGF-alpha), Bcl-2, caspase). CONCLUSION: In this unique autologous large-animal model, MSCs did not exhibit reparative or paracrine protective properties.

Apoptotic effects on B-cell chronic lymphocytic leukemia (B-CLL) cells of heterocyclic compounds isolated from Guttiferaes.

A series of 10 heterocyclic compounds purified from Allanblackia were tested on two B cell lines, ESKOL and EHEB, and on cells from B-CLL patients. Several molecules inhibited the proliferation of both cell lines and promoted apoptosis of B-CLL cells through different mechanisms, some of them elicited a dissipation of the mitochondrial transmembrane potential, other triggered caspase-3 activation and cleavage of the inducible nitric oxide synthase. Blood mononuclear cells and B-lymphocytes from healthy donors appeared less sensitive than B-CLL cells. These results indicate that these molecules may be of interest in the development of new therapies for B-CLL.

Malignant hematopoietic cells induce an increased expression of VEGFR-1 and VEGFR-3 on bone marrow endothelial cells via AKT and mTOR signalling pathways.

Angiogenesis plays a significant role in a variety of malignant hematologic diseases, and it is recognized that it has prognostic value. However, the cellular mechanisms by which malignant hematologic cells induce angiogenesis are not well understood. In order to investigate the role of cells from B-cell chronic lymphocytic leukemia (B-CLL) and multiple myeloma (MM) in angiogenesis on human bone marrow endothelial cells (HBMEC), we analyzed the impact of factors secreted by B-CLL cells and by MM cells on HBMEC capillary tube formation on matrigel. It was found that, in addition to the secretion of angiogenic factors VEGF and b-FGF by B-CLL and MM cells, MM cells (but not B-CLL cells) induced a dramatic increase in expression of VEGFR-1 and VEGFR-3 on human bone marrow endothelial cells (HBMEC). It would seem that this increase in VEGFR-3 occurred via the ERK and mTOR pathways, since their respective inhibitors U0126, LY294002 or rapamycin were responsible for a decrease of VEGFR-3. In response to MM cells-increased VEGF receptors on HBMEC, endothelial cell migration was enhanced in a wound artificially produced in a semi-confluent HBMEC culture, a phenomenon which was also down-regulated by the same inhibitors that reversed the increase in VEGF receptors. The present study suggests that, in addition to the classic angiogenic pathway, another mechanism related to an increased expression of VEGFRs on HBMEC might exist in malignant hematopoietic angiogenesis.

Pro-apoptotic properties of hyperforin in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

The effects of the hyperforin (HF), a natural phloroglucinol purified from Hypericum perforatum, were investigated ex vivo on leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). HF was found to promote apoptosis of B-CLL cells, as shown by time- and dose-dependent stimulation of phosphatidylserine externalization and DNA fragmentation, by disruption of the mitochondrial transmembrane potential, caspase-3 activation and cleavage of the caspase substrate PARP-1. Moreover, HF-induced downregulation of Bcl-2 and Mcl-1, two antiapoptotic proteins that control mitochondrial permeability. HF also downregulated two proteins which are overexpressed by B-CLL patients' cells, the cell cycle inhibitor p27kip1 through caspase-dependent cleavage into a p23 form, and the nitric oxid (NO) synthase of type 2 (inducible NO synthase). This latter was accompanied by reduction in the production of NO known to be antiapoptotic in B-CLL cells. Preventing effects of the general caspase inhibitor z-VAD-fmk indicated that HF-promoted apoptosis of B-CLL cells was mostly caspase dependent. Furthermore, normal B lymphocytes purified from healthy donors appeared less sensitive to HF-induced apoptosis than B-CLL cells. These results indicate that HF may be of interest in the development of new therapies for B-CLL based on the induction of apoptosis and combination with cell cycle-dependent antitumor drugs.

Valproic acid inhibits proliferation and induces apoptosis in acute myeloid leukemia cells expressing P-gp and MRP1.

The multidrug resistance (MDR) phenotype, induced by the overexpression of several ABC transporters or by antiapoptotic mechanisms, has been identified as the major cause of drug resistance in the treatment of patients with acute myeloid leukemia (AML). In this study, we have shown that valproic acid (VPA) (a histone deacetylase inhibitor) can inhibit the proliferation of both P-glycoprotein (P-gp)- and MDR-associated protein 1 (MRP1)-positive and -negative cells. VPA also induced apoptosis of P-gp-positive cells. VPA induced apoptosis in K562 cells led to decrease in Flip (FLICE/caspase-8 inhibitory protein) expression with Flip cleavage, which could not be observed in HL60 cells. In HL60/MRP cell line, which proved to be resistant to apoptosis by VPA, we observed an abnormal expression of apoptotic regulatory proteins, overexpression of Bcl-2 and absence of Bax. Also, the Bcl-2 antagonist HA14-1 rapidly restored apoptosis in this cell line. Cotreatment with cytosine arabinoside induced very strong apoptosis in both K562/DOX and HL60/DNR cell lines. VPA also induced apoptosis in AML patient cells expressing P-gp and/or MRP1. Our findings show VPA as an interesting drug that should be tested in clinical trials for overcoming the MDR phenotype in AML patients.

P-glycoprotein in blood CD4 cells of HIV-1-infected patients treated with protease inhibitors.

OBJECTIVE: Many factors are involved in the virological failure of antiretroviral treatments such as low pharmacological plasma levels of drugs, poor adherence to therapy and emergence of viral resistance. P-glycoprotein (P-gp) has been demonstrated to play a role in multidrug resistance in the therapy of solid tumours, haematological malignancies and Plasmodium falciparum infection. HIV-1 protease inhibitors (PIs) have been described to be substrates of P-gp. In vitro and in vivo studies performed in mice have demonstrated that P-gp may affect the oral bioavailability and intracellular accumulation of PIs. P-gps have been detected on peripheral CD4 blood cells in HIV-1-infected, but antiretroviral-naive patients. METHOD: We quantified P-gp expression and performed functional tests of P-gp activity in the CD4 cells in HIV-1-infected patients, with and without virological failure, treated with PIs, and in healthy patients (control group). RESULT: Out of the 18 HIV-infected patients studied, P-gp expression and function were found in the CD4 cells of six patients (four of 10 without, and two of eight with virological failure). Out of the 43 healthy patients studied, P-gp expression and function were found in the CD4 cells of 11 patients (26%). We found P-gp in peripheral CD4 cells of patients treated with PIs, with and without virological failure, within the same frequency than in antiretroviral naive patients or than in non HIV-infected patients. CONCLUSIONS: P-gp expression in peripheral CD4 blood cells does not seem to be enhanced by PI treatment and does not seem to be linked particularly to virological failures. These facts do not preclude of the role of P-gp on PI absorption or efficacy in other compartments of the body such as gut, lymph nodes or brain in HIV-1 PI-treated patients.

Effect of the multidrug inhibitor GG918 on drug sensitivity of human leukemic cells.

The drug GG918 has been specifically developed for overcoming MDR phenotype and is now in use in clinical trials. In this study, the effects of GG918 on leukemic cell were investigated using a 3 day MTT assay. Results showed that, in a highly resistant P-gp(+) leukemic cell line, 0.1 microM of GG918 gives rise to a 40-fold sensitization to daunorubicin (DNR) (residual resistance: 2.1), a 57-fold sensitization to mitoxantrone (residual resistance: 1.5), and a 3.3-fold sensitization to idarubicin (residual resistance: 2.9). When human AB serum was added to the incubation medium, 1 microM of GG918 was needed to observe the full P-gp modulation potency described above. The effect of 1 microM of GG918 was tested on 27 samples of poor prognosis acute leukemia (25 AML, two ALL). DNR sensitization (using the MTT assay) and modulation of rhodamine 123 uptake were monitored and used as criteria for comparing the in vitro modulation potency of this new compound to the potency of 10 microM of verapamil, which was used as reference. A good correlation (r = 0.8, P = 0.001) was observed between the results of the two tests. Eleven out of the 26 cases tested were MDR1(+) (42%), and showed a higher IC50 for DNR than the negative cases (861 +/- 1284 nM vs 187 +/- 246 nM, P = 0.05). GG918 was able to modulate the in vitro resistance to DNR in eight cases (seven MDR1(+), no MDR1(-), one non-tested). Verapamil did not increase DNR toxicity in four of these eight cases, but was more efficient in one other MDR1(+) case. In conclusion, the DNR sensitivity of the majority of the fresh AML samples expressing P-gp could be modulated in vitro by 1 microM of GG918.

Multicentric evaluation of the MDR phenotype in leukemia. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

The wide discrepancies in the frequency of 'positive' samples for multidrug resistance (MDR) phenotype within the same type of tumor observed in the literature justified the need for the definition of consensus recommendations. To define standard techniques of MDR phenotype measurement, we ran a large multicentric evaluation of the different methods available. Thirty-six French centers participated in the study, and 742 samples of 2-10 x 10(6) viable cells were sent by overnight express mail between December 1993 and February 1996. The same batches of MRK16, 4E3 and UIC2 were used. Nineteen samples of leukemia (12 AML, 1 ALL, 6 lymphoproliferative syndromes) and six leukemic cell lines with different levels of MDR expression were tested. Five meetings reached agreement concerning the guidelines for each technique, except immunocytochemistry. The 19 fresh samples were tested by each center using one to four techniques among cytofluorometry, immunocytochemistry, functional tests and RT-PCR. Five samples were diagnosed as 'negative' according to local criteria, with few discordant results (0 to 16% of 'positive' results). For all the 14 remaining samples, large discrepancies were observed from center to center, and from one technique to another. No correlations could be found between techniques. Flow cytometric analysis of cells already exposed to MRK16 or control IgG2A, fixed in paraformaldehyde and sent to centers did not reduce the discrepancies between centers in two of the four samples with moderate expression, emphasizing the role of histogram interpretation. The use of alternative monoclonal antibodies (4E3 and UIC2) did not reduce the discrepancies observed. In a second step, the K562 parental cell line, a low resistant subline (K562/HHT100, x7 resistance index to DNR) and a high resistant subline (K562/HHT300, x125 resistance index to DNR) were sent blindly three times, with an increasing level of recommendations for flow cytometry. Dramatic improvements were observed in cytometric results when the result was expressed as the ratio of arithmetic mean of fluorescence of antibody (10 microg of MRK16)/arithmetic mean of fluorescence of control (10 microg IgG2A): the proportion of expected results increased from 61 to 100% for K562, and from 37 to 85% for K562/HHT100. For uptake and drug efflux measurements, the use of 1 h uptake of 0.1 microM of rhodamine, followed by 1 h efflux +/-10 microM of verapamil, permitted an increased reproducibility of the technique from 71 to 100% for K562 and K562/HHT100. Whatever the technique used, concordant results were obtained for K562/HHT300. The immunocytochemistry, using several antibodies (MRK16, JSB1 and C219) gave many non-interpretable results (44%), due to a frequent high background and discordant results between antibodies in the same centers, and discordant conclusions between centers. The group does not recommend this technique for circulating tumoral cells.

French multicentric evaluation of mdr1 gene expression by RT-PCR in leukemia and solid tumours. Standardization of RT-PCR and preliminary comparisons between RT-PCR and immunohistochemistry in solid tumours. French Network of the Drug Resistance Intergroup, and Drug Resistance Network of Assistance Publique-Hôpitaux de Paris.

Since there is no consensus on the techniques for multidrug resistance (MDR) phenotype evaluation, many discrepancies concerning the importance and frequency of mdr1 gene expression in leukemias and solid tumors are observed in the literature. In order to establish an inter-laboratory consensus in France, a multicenter study was carried out to propose further guidelines for MDR phenotype evaluation. The techniques used by the 38 laboratories participating in the trial were: immunodetection (immunohisto and/or cytochemistry, flow cytometry), functional tests, reverse transcription-polymerase chain reaction (RT-PCR) or Northern blot. We present the results obtained by 19 laboratories concerning the measurement of mdr1 gene expression assessed by RT-PCR or Northern blot in: (1)19 samples of tumor cells obtained from leukemic patients; (2) six solid tumor samples obtained at surgery; (3) eight cell lines exhibiting variable levels of resistance, and; (4)10 preparations of RNA and of cDNA obtained from solid tumors. Standardization of the RT-PCR technique and preliminary results comparing RT-PCR with immunohistochemistry in solid tumors are also reported.

Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe.

Multidrug resistance (MDR) is characterized by a decrease in the efficiency of chemotherapeutic agents correlated with the expression and activity of a membrane protein: the permeability-glycoprotein (Pgp 170). Clinically, detection of MDR can be performed by functional tests based on the accumulation of fluorescent compounds such as rhodamine 123. With the aim of improving the sensitivity of such analysis, we have evaluated JC-1, a fluorescent lipophilic carbocyanine dye. Above a critical concentration, JC-1 aggregates in a 'liquid crystal' form. Aggregates display a specific red emission band centered at 597 nm whereas the monomers display a green emission band centered at 540 nm. JC-1 was avidly accumulated in sensitive K562 cells where it displayed both a green cytoplasmic and red mitochondrial fluorescence. In contrast, JC-1 was poorly accumulated in resistant K562 cells, which displayed only a slight green fluorescence. The level of JC-1 accumulation was correlated with the level of Pgp expression detected by MRK16 and UIC2 antibodies on a set of K562 subclones with increasing resistance levels. The specific fluorescence properties of JC-1 allow accurate discrimination between low-level resistant cells and sensitive cells. Chemosensitizers such as verapamil, cyclosporine A or S9788 restored JC-1 accumulation in resistant cells. The fluorescence properties of JC-1 could therefore be used for monitoring the effects of reversing agents.

Glutathione system, topoisomerase II level and multidrug resistance phenotype in acute myelogenous leukemia before treatment and at relapse.

In order to better understand acquired resistance to antitumor agents in acute myelogenous leukemia (AML), we investigated various drug resistance mechanisms; namely, topoisomerase II (topo II), glutathione system and P-glycoprotein (P-gp). Blast cells of 31 patients with AML, 21 before treatment (BT) and 10 at relapse (AR) were studied. Topo II was evaluated by Western blot analysis. Glutathione-S-transferase activity (GST) and glutathione content (GSH) were investigated by spectrophotometric assays. GST isoenzymes (-alpha, -mu and -pi) were tested by Western blot and by immunocytochemical staining. P-gp was evaluated by an immunocytochemical method using MRK 16 antibody. Our results showed that GST, GSH and GST-pi were similar in patients BT and AR GST-mu was detected in 13/21 AML BT and in 5/10 AML AR. GST-alpha expression was higher (p < 0.05) in AML AR (60 +/- 105 AU/mg) compared to AML BT (10 +/- 10 AU/mg). A relationship was found between GST-pi quantitation evaluated by Western blot and immunocytochemical staining, whereas no correlation was observed for the other isoenzymes. Topo II was detected in only 4 AML BT and 3 AML AR. Eleven out of 21 AML BT and 3/10 AML AR expressed P-gp with immunohistochemical study. These results indicate that only the "glutathione system", especially the GST-alpha could be involved in drug resistance in AML.

Drug resistance mechanisms in chronic lymphocytic leukemia.

Peripheral blood samples from 18 patients with chronic lymphocytic leukemias (CLL) who were either untreated but who were later sensitive to chlorambucil (CLL S) or resistant to a combination containing doxorubicin, vincristine, cyclophosphamide and prednisone (CLL R) were studied for glutathione system, P-glycoprotein, PCNA and topoisomerase II expression. P-glycoprotein expression detected by an immunocytochemical technique using MRK 16 antibody was present at the same level in CLL S and CLL R. The percentage of cells positive for P-gp was below 5% in all samples tested. Topoisomerase IIalpha level was quantified by Western blot analysis. None of the 18 CLL samples had detectable topoisomerase IIalpha protein. In addition, 12 CLL were tested for PCNA staining and no samples had more than 1% of positive cells at immunocytochemical detection indicating that CLL cells were not engaged in the cell cycle. Some differences were found between CLL S and CLL R in the glutathione system. Glutathione concentration (GSH) and GST activity was the same in CLL S and CLL R. The glutathione-S-transferase (GST) isoenzyme profile was different in the two CLL groups. The mean GST-pi and GST-alpha quantitation were twice as high as in CLL R compared to CLL S, but this difference did not reach statistical significance because of large variations between CLL samples. A significant correlation was observed between GST-pi expression and GST activity using CDNB as the substrate. GST-mu was detected in only one of seven CLL before therapy and in six of 11 resistant to chemotherapy. No correlation was found between P-glycoprotein expression, GST activity and the different GST isoenzymes studied. These results suggest that the glutathione system could play a role in the resistance of anticancer agents in chronic lymphocytic leukemia. The role of the other drug resistance mechanisms (P-glycoprotein and topoisomerase IIalpha) seems to be of limited importance.

Reversal of multidrug resistance by SDZ PSC 833, combined with VAD (vincristine, doxorubicin, dexamethasone) in refractory multiple myeloma. A phase I study.

SDZ PSC 833, a non-immunosuppressive cyclosporin analogue reverses multidrug resistance (MDR) in vitro by inhibiting P-glycoprotein (P-gp) mediated drug efflux. We performed a dose escalation study of SDZ PSC 833 combined with VAD chemotherapy in refractory multiple myeloma (MM). Twenty-two MM patients who were refractory to doxorubicin/vincristine/dexamethasone (VADr, n=11) or had failed multiple regimens (n=6) or were melphalan-refractory (MELr, n=5), were treated with one to three cycles of VAD combined with oral SDZ PSC 833, which was administered at escalating dosages starting at 5 mg/kg/day to 15 mg/kg/day for 7 days. The median trough and peak blood levels of SDZ PSC 833 ranged from 461/1134 ng/ml at 5 mg/kg/day to 821/2663 ng/ml at 15 mg/kg, respectively. With addition of SDZ PSC 833 (5 mg/kg) the mean plasma AUC 0-->96 h of doxorubicin as compared with control patients treated with VAD increased from 779 to 1510 ng/ml/h (P=0.0071), while the doxorubicin clearance was reduced from 47.6 to 27.8 l/h/m2 (P=0.0002). The clearance of doxorubicinol was reduced accordingly. Because of the increased plasma AUC, the dose of doxorubicin and vincristine had to be reduced in 13 patients to 50% (n=1) or 75% (n=12). A further dose-escalation of SDZ PSC 833 did not lead to a proportional increase of doxorubicin AUC. Toxicity WHO CTC grade 2 or 3 included hypoplasia (18/22), constipation (10/22), hyponatremia (3/22) and infections (6/22). A partial response or stable disease was achieved in eight and six patients, respectively. In 17 evaluable patients the mean percentage of pretreatment bone marrow plasma cells which expressed P-glycoprotein was 40%. The pretreatment in vitro rhodamin retention in CD38++ myeloma cells was reversible by 2 microM SDZ PSC 833 with 15-98% in 7/9 tested patients. In 4/5 responding patients analyzed before and after treatment with VAD + SDZ PSC 833, a reduction of P-gp + plasma cells was observed. It is concluded, that the blood concentrations of SDZ PSC 833 attained in MM patients increase with dose after oral administration. It can be safely combined with VAD chemotherapy. SDZ PSC 833 diminishes the clearance of doxorubicin, leading to an increase of the plasma AUC of doxorubicin. In addition, it is an effective inhibitor of P-gp mediated efflux of doxorubicin in myeloma tumor cells in vitro. Therefore, a proportional dose-reduction of doxorubicin and vincristine is warranted. Phase II/III studies in refractory MM are in progress to evaluate the therapeutic efficacy of SDZ PSC 833 with VAD.

[Multicentric evaluation of MDR phenotype in leukemia: intermediate analysis of the French study]. / Evaluation multicentrique du phénotype MDR dans les leucémies: analyse intermédiaire de l'expérience française.

Thirty-six French centres are involved in an evaluation of the techniques used for MDR phenotype measurement. Until now, 14 samples of various kinds of leukemia (mainly acute myelogenous leukemia) and three cell lines with different levels of resistance were sent by one centre and tested. MRK16 antibody was used for flow cytometry and immunocytochemistry, RNA was measured by RT-PCR, rhodamine or anthracyclin efflux were tested for functional assay. Wide discrepancies were observed in the results, mainly with flow cytometry, specially for the samples with a probable low level of MDR1 expression. The importance of histogram interpretation was documented by the comparative analysis of results obtained on cells already marked with MRK16, fixed and sent to all centers. The use of the ratio of the mean of fluorescence, instead of percentage, should help for standardization. The use of only one control RNA (used at different dilutions) for standardisation of RT-PCR could help in decreasing the discrepancies observed. The mean of fluorescence should also be used for expressing the rhodamine cell content.

Sequential emergence of MRP- and MDR1-gene over-expression as well as MDR1-gene translocation in homoharringtonine-selected K562 human leukemia cell lines.

To investigate the mechanism of resistance to an antineoplastic natural product homoharringtonine (HHT) in leukemic cells, we have established 5 sub-lines of human myeloid leukemia K562 cells, designated as K-H30, K-H100, K-H200, K-H300 and K-H400, which showed progressive resistance to different concentrations of HHT. These sub-lines were cross-resistant to daunorubicin, vincristine, etoposide and mitoxantrone, but not to melphalan. Immunofluorescence with monoclonal anti-Pgp antibody MRK16 and Northern-blot analysis demonstrated that resistance to HHT is related to the sequential emergence of MRP- and MDR1-gene over-expression. In the low-level-resistant K-H30 sub-line, the MDR1 gene was not over-expressed, but the MRP gene was over-expressed 2.1-fold. In the intermediate-level-resistant K-H100 and K-H200 sublines, both the MRP and the MDR1 genes were over-expressed. However, in the high-level-resistant K-H300 and K-H400 sublines, MDR1-gene over-expression predominated (20- and 21-fold respectively). On the other hand, GST pi-gene expression was decreased in all 5 sub-lines. Southern-blot analysis revealed no MRP-gene amplification in any of the 5 sub-lines, whereas the MDR1 gene was amplified in the high-level-resistant K-H300 and K-H400 sub-lines. The most interesting observation is a homogeneously staining region (HSR) found in chromosome 2 of the K-H300 and K-H400 sub-lines. Chromosome painting and in situ hybridization demonstrated that this HSR was translocated from chromosome 7 and consisted of the amplified MDR1 gene, suggesting that there is a relationship between MDR1-gene, translocation and MDR1-gene amplification.

Glutathione-S-transferases pi, alpha, mu and mdr1 mRNA expression in normal lymphocytes and chronic lymphocytic leukemia.

Chronic B cell lymphoproliferative disorders are frequently sensitive to alkylating agents. To assess the glutathione-S-transferases (GSTs) gene expression in B tumoral lymphocytes, possibly responsible for this sensitivity, we developed a sensitive RT-PCR assay for the three isoenzymes GST pi, GST mu and GST alpha mRNA. Normal B and T lymphocytes from 11 blood donors were separated by magnetic beads and tested with this assay. The GST pi was the most abundant transferase, and was detected in all B and T cell samples. GST mu was undetectable ('null' phenotype) in 6/11 normal donors, either in B or T cells. GST alpha was very stable from donor to donor, and was highly correlated between B and T cells of the same individual (P < 0.0001). There is no correlation between the three isoenzymes, and between each isoenzyme and mdr1 gene expression. Twenty-three B lymphoproliferative disorders (20 B-CLL, 3 CD5- chronic lymphoproliferative syndromes) were tested with the same technique. An average decrease of 57% of the GST pi expression was noted in the mononuclear cells of these patients (P < 0.02), with no differences between the untreated and treated cases. The GST alpha and mdr1 mRNA levels did not differ from normal B lymphocytes, but the proportion of patients with no detectable expression of GST mu is lower than in the control (13%). Interestingly, the low content of GST pi in B-CLL could explain the frequent sensitivity of this disease to alkylating agents.

Overexpression of cyclin D2 in chronic B-cell malignancies.

Tumor progression in B-cell chronic lymphocytic leukemia (B-CLL) is thought to result from the gradual accumulation of small resting G0/G1 phase lymphoid cells rather than the proliferation of actively dividing cells. The recent identification of G1 cyclins that are likely to control both the progression through G0 and G1 phase and the G1/S transition prompted us to study the mRNA expression of D-type cyclins in the peripheral blood lymphocytes from 34 patients with B-CLL, 7 patients with lymphoplasmacytic lymphoma (LPL), and 2 patients with mantle cell lymphoma (MCL). Cyclin D2 mRNA was, on average, 5- to 10-fold overexpressed in most of the samples studied (B-CLL, 29/34; LPL, 7/7; MCL, 0/2) as compared with normal resting B lymphocytes, in which cyclin D2 mRNA was barely detectable. In situ hybridization with cyclin D2 digoxigenin-labeled mRNA probe showed that all the cells from a given sample were stained with approximately the same intensity. Cyclin D3 was never detected in any of the samples tested, whereas cyclin D1 was expressed in only the 3 cases (1 LPL and 2 MCL) bearing a t(11;14) translocation. A trisomy 12 was found in 4 of 19 (21%) B-CLL or LPL cases for which cytogenetic analysis was available. Although the cyclin D2 gene has been mapped to chromosome 12p13, there was no apparent correlation between trisomy 12 and the level of cyclin D2 expression. Cell cycle analysis by flow cytometry after staining with propidium iodide consistently showed that more than 96% of the cells were in G0/G1 phase, whatever the importance of cyclin D2 overexpression was, and that cyclin D2 overexpression in B-CLL was not associated with any modifications of the cell cycle repartition. No consistent overexpression of cyclin D2 was found in acute myeloid leukemias. In conclusion, overexpression of cyclin D2 mRNA was found to be an almost constant feature in B-CLL and LPL. Therefore, it led us to hypothesize, with the support of data from some transfection experiments previously reported in murine hematopoietic cell lines, that cyclin D2 might play a role in B-CLL pathogenesis, possibly by preventing cells from programmed cell death.

Over-expression of cyclin D1 in chronic B-cell malignancies with abnormality of chromosome 11q13.

Accurate identification of B-cell chronic malignancies is sometimes uncertain, despite careful cytologic and immunophenotypic evaluation. Cytogenetics and molecular biology studies may therefore prove useful, because some of these disorders are associated with non-random abnormalities, such as the t(11;14)(q13;q32) translocation and bcl-1 rearrangement mainly observed in mantle-cell lymphoma (MCL). We studied the expression of cyclin D1 in malignant lymphoid cells from the peripheral blood of 32 patients with various B-cell chronic lymphoproliferative disorders, using Northern blot (NB) and RNA in situ hybridization (ISH). Cytogenetic analysis was informative in 18 cases, and most of the missing karyotype data were from typical B-CLL cases where a t(11;14) is unlikely to be found. Over-expression of cyclin D1 mRNA was observed by both NB and ISH in four samples (MCL; two cases; lymphoplasmacytic lymphoma: one case, unclassified B-cell chronic disorder: one case). In each of these cases there was an abnormality of chromosome 11q13, either a t(11;14)(q13;q32) translocation (three cases) or a del(11)(q13) without evidence of chromosome 14 involvement (one case). Cytogenetic and gene rearrangement studies are not available in all institutions and have some technical pitfalls. Because of its close association with bcl-1 rearrangement and/or t(11;14), the demonstration of cyclin D1 mRNA over-expression either by NB, or, more conveniently, by ISH, may represent additional information which could be of help for the identification of B-cell malignancies.

Evaluation of the clinical relevance of the anionic glutathione-s-transferase (GST pi) and multidrug resistance (mdr-1) gene coexpression in leukemias and lymphomas.

By using RNA slot-blot technique, the frequency and the degree of GST pi and mdr-1 gene coexpression were investigated in 23 AML patients, 9 ALL, 9 CLL and 11 cases of NHL in an attempt to study their clinical and prognostic relevance. GST pi and mdr-1 levels were expressed as arbitrary units (U) with respect to the negative controls (U = 0), MCF7 and HL60 sensitive cell lines, and the positive controls (U = 10), MCF7/DOXO and HL60/DNR resistant cell lines. The concomitant GST pi/mdr-1 gene overexpression showed a negative prognostic value in the set of newly diagnosed AML pts (10 cases), furthermore higher GST pi and mdr-1 mRNA levels were averagely detected in the relapsed/resistant ALL pts (4 cases), and in CLL (7 cases) and NHL (8 cases) heavily pretreated patients who were unresponsive to chemotherapy and with a disease progression. These preliminary data show that two different mechanisms of drug resistance can be coexpressed at the same time in those leukemias and lymphomas with a clinically unfavourable course.