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Keywords:

  • leptin receptor;
  • leukaemia;
  • differentiation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

The leptin receptor is a member of the cytokine receptor superfamily, and is expressed in CD34 haemopoietic stem cells. We examined expression of the leptin receptor in fresh human leukaemia cells. Northern blot analysis showed the leptin receptor was expressed in leukaemic cells from patients with acute myeloblastic leukaemia, acute lymphoblastic leukaemia and chronic myeloid leukaemia (CML). In CML, higher expression was observed in blast crisis than in chronic phase. The expression of leptin receptor decreased during in vitro differentiation of leukaemic blast cells. It appeared that expression of the leptin receptor was associated with immature leukaemic blast cells. Our findings may indicate the possibility that leptin has some role in leukaemia.

Haemopoiesis is regulated by a large number of cytokines. These cytokines exert their effects by binding to the specific receptors that are members of the cytokine receptor family. Leptin, the product of the ob gene, is a 16 kD secreted protein which regulates the size of the adipose tissue mass. The severe obese phenotype is caused by the inability to produce leptin in ob/ob mice ( Zhang et al, 1994 ). Tartaglia et al (1995 ) have isolated cDNAs encoding murine and human leptin receptor. Leptin receptor is a single membrane-spanning receptor which is closely related to the gp130 signal transducing component of the interleukin-6 (IL-6) type cytokine receptors. Leptin receptor has several alternatively spliced isoforms with different length cytoplasmic domains ( Tartaglia et al, 1995 ; Cioffi et al, 1996 ). In db/db mice the profound obese phenotype is caused by a mutation leading to the production of an aberrant splice product of this isoform ( Chen et al, 1996 ). Recently it has been reported that leptin receptor is expressed in CD34-positive haemopoietic stem cells ( Cioffi et al, 1996 ). The proliferation and maturation of normal haemopoietic cells are positively or negatively regulated by a variety of haemopoietic growth factors. These effects are mediated through the high-affinity binding of extracellular factors to specific cell surface receptors. In addition to normal haemopoietic cells, leukaemic cells have been shown to express functional receptors for a variety of growth factors such as interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) ( Park et al, 1989 ; Begley et al, 1987 ; Dubreuil et al, 1988 ; Ikeda et al, 1991 ). Several investigators have shown that disregulated expression of growth factors and/or their receptors could be involved in the pathogenesis of leukaemias ( Depper et al, 1984 ; Rambaldi et al, 1988 ; Mitjavila et al, 1991 ; Furitsu et al, 1993 ; Matsumura et al, 1995 ). Therefore it is important to know the expression of leptin receptor in leukaemic blast cells. In the present study we have examined the expression of leptin receptor in blast cells of patients with leukaemias.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Growth factors

Recombinant human GM-CSF, G-CSF and IL-3 were kindly provided by Kirin Brewery Co. Recombinant SCF was kindly provided by Kirin-Amgen Inc. Recombinant human leptin was purchased from R & D System Inc. Human leptin was iodinated by the chloramine-T method with a minor modification as described elsewhere ( Fraker & Speck, 1978). Briefly, leptin (5 μg), 18.5 Mbq of Na125I, and chloramine-T (3.75 μg/ml) in 30 μl of 0.3 M sodium phosphate (pH 7.4) were placed in a 1.5 ml polypropylene tube. The iodination reaction was continued for 10 min at 22°C and stopped by addition of 5 μl of sodium metabisulphite (60 μg/ml). Labelled leptin was separated from free 125I by column chromatography using a Sephadex G-25 (Pharmacia Fine Chemicals, Uppsala, Sweden).

Cells and cell culture

Human leukaemia cell lines K562, HEL, HL60, U937, THP-1, KG-1, MOLT4 and CCRF-CEM were obtained from Health Science Research Resources Bank (Osaka, Japan). Cells were maintained in RPMI-1640 medium containing 10% fetal calf serum (GIBCO BRL, Grand Island, N.Y.) at 37°C in a 95% air/5% CO2 humidified incubator. MO7E, kindly provided by Dr Matsumura, Osaka University, was maintained in RPMI-1640 medium containing 10% fetal calf serum and 5 ng/ml human GM-CSF.

CD34-positive stem cells were obtained from patients with malignant lymphoma recovering from consolidation chemotherapy (CHOP) in complete remission state. High-purity CD34-positive cells (98.8%) could be selected from peripheral blood leukapheresis samples by an immunomagnetic beads method as previously reported ( Hasuike et al, 1997 ). Bone marrow cells were obtained from healthy donors by aspiration.

Leukaemic blast cells were obtained from patients with leukaemia. The diagnosis was based on cell morphology and genetic markers. The subtype and numbers of cases were 28 cases of acute myeloblastic leukaemia (AML) [M0 (n = 2), M1 (n = 5), M2 (n = 7), M3 (n = 5), M4 (n = 3), M5 (n = 2), M6 (n = 4)], 11 cases of acute lymphoblastic leukaemia (ALL) and eight cases of chronic myeloid leukaemia (CML) of which four cases were in myeloid blast crisis.

Mononuclear cells were prepared from heparinized fresh peripheral blood or bone marrow aspiration samples by density gradient centrifugation on Ficol-Conrey (density = 1.077 g/ml). All samples were taken with informed consent.

Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis

Total cellular RNA was extracted by the acid guanidium–thiocyanate phenol–chloroform method ( Chomczynski & Sacchi, 1987). cDNA was synthesized from 1 μg of RNA with 200 U of reverse transcriptase in 1 × RT reaction buffer containing 100 pmol of random hexamer primers, 1 mmol/l dNTPs and 20 U of RNasin at 37°C for 1 h. For amplification of the cDNA products, human leptin receptor specific primers (5′-TGTTGTGAATGTCTTGTGCC-3′ and 5′-CACTCACAACATCATACTGG-3′) or human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers (5′-CCATGGAGAAGGCTGGGG-3′ and 5′-CAAAGTTGTCATGGATGGATGACC-3′) and 0.5 U AmpliTaq were added in 1 × PCR reaction buffer. PCR was performed on the GeneAmp PCR System 9600 for 35 cycles. Each cycle was carried out at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Amplified fragment was cloned into TA cloning vector and identified by DNA sequencing.

Northern blot analysis

Total RNA (10 μg) was separated by electrophoresis on 1% agarose gel containing formaldehyde, transferred to nylon membranes (Hybond-N) by capillary action, and fixed. Human leptin receptor or rat GAPDH cDNA was labelled with [α-32P]dCTP using hexadeoxynucleotide random primers. The membranes were hybridized with 32P-labelled leptin receptor or GAPDH cDNA as probes in 50% formamide, 3 × SSC (1 × SSC, 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.4), 50 m M Tris-HCl (pH 7.5), 0.1% sodium dodecyl sulphate (SDS), 20 μg/ml of tRNA, 20 mg/ml of boiled salmon sperm DNA, 1 m M EDTA, and 1 × Denhardt (0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone, and 0.02% Ficoll) for 40 h at 37°C. The nylon membranes were washed with 2 × SSC, 1% SDS, and 1 × Denhardt at 37°C for 1 h, followed by 0.1 × SSC and 1% SDS at 50°C for 1 h and then underwent autoradiography using intensifying screens at −80°C

Assay for binding of 125I-leptin

The cells were incubated with 125I-leptin (62 000–67 000 cpm/ng) in α-medium containing 0.1% bovine serum albumin, 20 m M HEPES, and 0.02% sodium azide (pH 7.4) at 15°C for 60 min. The cells were separated from unbound 125I-leptin by centrifugation on a cushion of di-n-butyl phthalate. The radioactivity associated with the pellets was counted in an automatic gamma counter. Specific binding was determined by subtracting the nonspecific binding measured in the presence of 100-fold excess unlabelled leptin from total binding.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Expression of leptin receptor mRNA was examined by means of RT-PCR and Northern blot analysis in normal bone marrow cells, CD34-positive haemopoietic stem cells and various types of human leukaemia cell lines. Fig 1 shows RT-PCR analysis for expression of leptin receptor mRNA. Leptin receptor was detected in normal bone marrow, CD34-positive haemopoietic stem cells and almost all the human leukaemia cell lines examined, except for CCRF-CEM. As shown in Fig 3, overexpression of leptin receptor was observed in K562, HEL and MO7E cells by Northern blot analysis. In accordance with the previous findings on human leukaemia cell lines, leptin receptor transcripts were detected in both short and long forms. Fresh leukaemic cells from AML, ALL and CML patients were examined for leptin receptor gene expression by Northern blot analysis. In some cases of AML and ALL, constitutive high expression of leptin receptor mRNA was detected ( Figs 2a and 2b). There was no significant correlation between the level of leptin receptor expression and the French–American–British (FAB) classification. For CML, higher expression was observed in the cases of blast crisis than those in chronic phase. Similarly, in the patient CML8, higher expression of leptin receptor was observed in blast than in chronic phase (Fig 4, lanes 10 and 11). To investigate whether expression of leptin receptor is associated with the cell differentiation, we cultured isolated CD34 positive haemopoietic stem cell for 14 d with SCF, IL-3, GM-CSF and G-CSF as described elsewhere ( Sakai et al, 1997 ), then examined the expression by Northern blot analysis. Leptin receptor gene expression decreased in the differentiated cells (Fig 5, lanes 8, 9 and 10). Similarly, we cultured the fresh blast cells of the patient CML6 (a patient with CML blast crisis) with G-CSF for 21 d, then almost all cells were differentiated to morphologically mature cells. Northern blot analysis indicated the expression of leptin receptor dramatically decreased in the differentiated cells (Fig 5, lanes 11 and 12).

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Figure 1. 0) were amplified using specific primers for human leptin receptor and human GAPDH primers.

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Figure 3. 4-positive cells (lane 9) and bone marrow cells (lane 10) were electrophoresed, transferred and hybridized with the human leptin receptor cDNA and rat GAPDH cDNA.

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Figure 2. : ALL8 (PB); 13: ALL9 (PB); 14: ALL10 (BM); 15: ALL11(BM).

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Figure 4. (CP); 7: CML5 (BC); 8: CML6 (BC); 9: CML7 (BC); 10: CML8 (CP); 11: CML8 (BC).

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Figure 5. : neutrophils from healthy volunteer 1, 6: neutrophils from healthy volunteer 2; 7: peripheral blood leukapheresis sample from patient with malignant lymphoma; 8: isolated CD34- positive cells; 9: differentiated cells from the isolated CD34-positive cells cultured with SCF, IL-3, GM-CSF and G-CSF; 10: the differentiated cells from the isolated CD34-positive cells cultured with G-CSF; 11: blast cells of CML6 (CML-BC); 12: the cultured differentiated blast cells of CML6 (CML-BC).

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To determine whether leptin may have affinity to the cell surface leptin receptor, the binding study was carried out using 125I-labelled leptin. As shown in Fig 6, specific binding was observed in K562, HEL and MO7E cell lines. In the fresh leukaemic blast cells from the patient CML6 (CML-blastic crisis), specific binding was also obtained.

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Figure 6. 2 cells was almost saturated (data not shown). Specific binding was determined by subtracting the nonspecific binding measured in the presence of 100-fold excess unlabelled leptin from total binding. Each data is the mean of duplicate determinations.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

In the present study we have shown that leptin receptor was constitutively expressed in human fresh leukaemic cells from AML, ALL and CML patients. We did not observe any obvious correlation between expression of leptin receptors and FAB classification. However, in patients with CML the expression levels of leptin receptor were clearly associated with the clinical phase. Higher expression of leptin receptor was observed in blast crisis than in chronic phase. As the blast crisis developed, the percentage of immature blasts and promyelocytes increased. The expression of leptin receptor decreased during in vitro differentiation of leukaemic blast cells. Therefore, it appeared that expression of the leptin receptor was associated with immature leukaemic blast cells in CML. Similarly, higher expression of leptin receptor was observed in CD34-positive immature haemopoietic cells than in mature neutrophils (Fig 5). Recently, Gainsford et al (1996 ) have reported that leptin receptor was expressed in immature granulocytes but less in mature granulocytes. Mikhail et al (1997 ) have reported that leptin had a significant effect on haemopoietic development, and Umemoto et al (1997 ) have reported that leptin stimulated the proliferation of murine myelocytic and primitive haemopoietic progenitor cells. Leptin may play an important role in early haemopoiesis as a haemopoietic growth factor. Several haemopoietic growth factors stimulate the proliferation of leukaemic cells which have the specific receptors. Our findings and those of others may indicate the possibility that leptin stimulates leukaemic cell growth. Bone marrow contains many adipocytes that are a natural source of leptin. Therefore it may be possible that leptin plays an important role in leukaemic cell proliferation in bone marrow. To clarify this possibility, we also examined the effect of leptin on proliferation or differentiation of fresh leukaemic cells using the MTT assay or morphological study, respectively. However, we did not find any effects of leptin on leukaemic cells (data not shown). The co-effects of leptin with other cytokines has not been discussed here; this possibility will be addressed in further investigations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

This work has been supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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