Phytohemagglutinin-induced IL2 mRNA in whole blood can predict bortezomib-induced peripheral neuropathy for multiple myeloma patients.

The proteasome inhibitor bortezomib has revolutionized the treatment of multiple myeloma. However, bortezomib-induced peripheral neuropathy (BiPN) is a serious complication that compromises clinical outcome. If patients with a risk of developing BiPN could be predicted, physicians might prefer weekly, reduced-dose, or subcutaneous approaches. To seek biomarkers for BiPN, we conducted a multicenter prospective study using a simple and unique system. Multiple myeloma patients received twice-weekly or weekly 1.3 mg/m(2) bortezomib intravenously, and a 2-ml sample of whole blood was obtained before treatment and 2-3 days and 1-3 weeks after the first dose. Induction of gene expression was then quantified by real-time PCR. Of a total of 64 enrolled patients, 53 patient samples qualified for mRNA analysis. The BiPN grade was associated with phytohemagglutinin-induced IL2, IFNG and TNFSF2, as well as with lipopolysaccharide-induced IL6 levels. More importantly, of the 19 patients showing a 3-fold increase in phytohemagglutinin-induced IL2, 14 did not suffer from BiPN (73.7% prediction), whereas of the 34 patients with a <3-fold increase, 23 experienced BiPN (67.6% prediction). Therefore, we concluded that pretreatment of phytohemagglutinin-induced IL2 mRNA levels in whole blood serve as a promising biomarker for predicting BiPN, and this finding warrants validation in a larger study.

New highly potent and specific E6 and E7 siRNAs for treatment of HPV16 positive cervical cancer.

Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 the most prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with HPV16+ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16+ and HPV16-cells. Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of HPV16+ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21(WAF1/CIP1), as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected siRNAs was confirmed by retarded tumor growth of HPV16+ cells in NOD/SCID mice when locally injected in a complex with atelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related cancer.

Establishment of a new human acute monocytic leukemia cell line TZ-1 with t(1;11)(p32;q23) and fusion gene MLL-EPS15.

Human leukemia cell lines are of great value in investigating basic and applied aspects of cell biology and clinical medicine. There have been 37 leukemia cell lines carrying 11q23 translocation and MLL rearrangements; however, cell lines harboring with t(1;11)(p32;q23) have not been established. We report here for the first time a new acute monocytic leukemia (AMoL) cell line with t(1;11)(p32;q23), designated TZ-1, and herein describe its biological characteristics. Mononuclear cells isolated from the ascites from a patient with AMoL (French-American-British classification; acute myeloid leukemia M5a) were isolated and passaged by liquid culture medium for a year. TZ-1 cells revealed typical monocytic features in morphology and had a t(1;11)(p32;q23) translocation. The immunoprofiling as determined by flow cytometry showed that TZ-1 cells are positive for myeloid and monocytic markers with lymphoid-associated markers. Fluorescence in situ hybridization and reverse transcription-polymerase chain reaction analyses revealed MLL-EPS15 fusion transcript and protein. Taken together, these results suggest that TZ-1 is a new monocytic leukemia cell line with t(1;11) translocation and fusion gene MLL-EPS15. The established cell line, TZ-1, could provide a valuable model in the analysis of the pathogenesis of MLL-EPS15-positive leukemia and in the development of new agents for this type of leukemia.

Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA.

Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.

Expression and sequence analyses of p33(ING1) gene in myeloid leukemia.

p33(ING1) is a novel candidate tumor suppressor gene which is involved in the regulation of apoptosis. p33(ING1) interacts with p53 signaling pathway and regulates cellular growth. It has reported that the expression of p33(ING1) mRNA was decreased in lymphoid malignancies. We thus investigated the potential involvement of p33(ING1) abnormalities in myeloid leukemias. However, the levels of p33(ING1) transcript were almost equal in 3 AML cell lines and 10 fresh AML samples. In addition, neither point mutations nor deletions in p33(ING1) gene were found in myeloid leukemias. These results suggest that p33(ING1) may not be a major candidate tumor suppressor gene in myeloid leukemias.

Capsaicin-like anti-obese activities of evodiamine from fruits of Evodia rutaecarpa, a vanilloid receptor agonist.

Evodiamine, a major alkaloidal principle of Evodia fruits (Evodia rutaecarpa, Rutaceae), showed vanilloid receptor agonistic activities comparable to capsaicin. The Chinese literature refers to Evodia fruits as a "hot nature" herb. In spite of the similarities in the actions of evodiamine and capsaicin in vitro, evodiamine has no perceptible taste, including a peppery hot taste. Therefore, the effectiveness of evodiamine and the extract of Evodia fruits in preventing obesity on male C3H mice, or male SD rats were examined. When evodiamine was supplemented at 0.03% of the diet and fed to mice for 12 days, the perirenal fat weight became significantly lower than in the control group. The epididymal fat mass was also decreased in the evodiamine diet group. When evodiamine was supplemented at 0.02% in the form of ethanol extract of Evodia fruits to the high-fat diet and fed to rats for 21 days, the body weight, the perirenal fat weight, epididymal fat weight, the levels of serum free fatty acid, total lipids in the liver, triglyceride in the liver, and cholesterol level in the liver were significantly reduced as compared with the control diet group. Furthermore, both lipolytic activity in the perirenal fat tissue and specific GDP binding in brown adipose tissue mitochondria, as the biological index of enhanced heat production, were significantly increased in the evodiamine fed rats. Fasting mice subcutaneously administered 1-3 mg/kg evodiamine showed decreased core body temperature by 1-2 degrees C. This hypothermic effect was prevented by the pretreatment of intraperitoneally administered 10 mg/kg capsazepine, a vanilloid receptor antagonist. On the other hand, food-sated mice subcutaneously administered 1-3 mg/kg evodiamine showed unchanged core body temperature and increased tail skin temperature by more than 5 degrees C, suggesting the increased energy expenditure by enhanced heat dissipation. In conclusion, we have demonstrated that a novel non-pungent vanilloid receptor agonist, evodiamine, mimics the characteristic anti-obese effects induced by capsaicin. Evodiamine would induce heat loss and heat production at the same time and dissipate food energy, preventing the accumulation of perivisceral fat and the body weight increase.

A novel differentiation-inducing therapy for acute promyelocytic leukemia with a combination of arsenic trioxide and GM-CSF.

Arsenic trioxide (As2O3) effectively induces clinical remission via apoptosis in relapsed acute promyelocytic leukemia (APL). However, because this new anti-leukemic drug is also considered to be a poison, its possible adverse effects are a highly important issue related to its clinical use. We here investigated, both in vitro and in vivo, the effects of a combination of As2O3 and GM-CSF as a novel therapeutic approach for the treatment of APL. Treatment of both retinoic acid (RA)-sensitive and -resistant APL cell lines (NB4 and UF-1 cells, respectively), as well as primary APL cells with a combination of As2O3 and GM-CSF for 4 days resulted in inducing differentiation, but not apoptosis, to mature granulocytes. In addition, a combination of both agents induced degradation of the PML/RARalpha protein. GM-CSF was found to be associated with increased tyrosine phosphorylation of Jak2 kinase in both NB4 and UF-1 cells, and a specific inhibitor of Jak2, AG490, completely blocked the ability of GM-CSF to prevent apoptosis and induce differentiation of As2O3-treated UF-1 cells. In in vivo analysis, As2O3 induced differentiation of APL cells in a RA-resistant APL model of human GM-CSF-producing transgenic SCID mice that had a high level of human GM-CSF in their sera. In contrast, As2O3 alone diminished tumors in UF-1 cells transplanted into NOD/SCID mice via induction of apoptosis. In conclusion, a combination of As2O3 and GM-CSF appears to be a novel differentiation-inducing therapy in patients with APL, including relapsed or RA-resistant cases.

Novel mutation in the PML/RARalpha chimeric gene exhibits dramatically decreased ligand-binding activity and confers acquired resistance to retinoic acid in acute promyelocytic leukemia.

OBJECTIVE: All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood. MATERIALS AND METHODS: We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient. RESULTS: A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding. CONCLUSIONS: These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.

Activation of the p21(CIP1/WAF1) promoter by bone morphogenetic protein-2 in mouse B lineage cells.

BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21(CIP1/WAF1) expression. In the present study, we demonstrated that BMP-2 activated the mouse p21(CIP1/WAF1) promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter (-1928/-1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells. These results suggested that BMP-2 might activate p21(CIP1/WAF1) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.

Mcl-1, an early-induction molecule, modulates activin A-induced apoptosis and differentiation of CML cells.

Activin A, one member of the transforming growth factor (TGF)-beta superfamily, is known to be a commitment factor for cell death and differentiation. In the present study, we demonstrate that human chronic myeloid leukemia (CML) cell lines, KU812 and K562 cells, either induced apoptosis or differentiation, respectively, by treatment with activin A. During these cell fate decisive events caused by activin A, rapid and transient up-regulation of Mcl-1 was observed in both cell lines. In activin A-induced apoptosis of KU812 cells, continuous up-regulation of Bax was observed. After the decrease in Mcl-1 expression had occurred, activation of caspase-9 and caspase-3 and cleavage of DFF45 were shown to take place in KU812 cells, resulting in the fragmentation of the genomic DNA of the cells. In contrast, the down-regulation of Mcl-1 without up-regulation of Bax caused accumulation of hemoglobin (Hb) contents in activin A-treated K562 cells. Interestingly, erythropoietin (EPO) prevented activin A-induced apoptosis with continuous expression of Mcl-1 and caused KU812 cells to undergo erythroid differentiation. To address the role of Mcl-1 in activin A-treated CML cells, KU812 and K562 cells were stably transfected with cDNA encoding Mcl-1 (designated as KU812/mcl and K562/mcl cells). As in combined effect of activin A and EPO on the parental KU812 cells, activin A induced differentiation, but not apoptosis, of KU812/mcl cells without modulating Bax levels. Activin A-treated K562/mcl cells, as well as parental cells, were only differentiated to erythroid cells. These results suggest that Mcl-1 is an early inducible gene activated by the activin A signaling pathway for both cellular differentiation and apoptosis, and continuous expression of Mcl-1 may be contributed to differentiation signals to the erythroid lineage in CML cells.

Quantitative monitoring of the PRAME gene for the detection of minimal residual disease in leukaemia.

PRAME (Preferentially expressed antigen of melanoma) has been previously identified as a melanoma antigen recognized by cytotoxic T cells (CTLs) and found to be expressed in a variety of cancer cells including leukaemic cells. We have screened 98 Japanese patients with leukaemia and lymphoma for expression of the PRAME gene using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Forty-one patients (42%) showed high levels of PRAME expression. Eight of these patients were then monitored using real-time PCR for a period of 10-37 months. Significant reductions in the PRAME expression were observed in all patients after chemotherapy. An increased expression was detected in the two patients who relapsed, one of which was before cytological diagnosis. These changes were correlated with those of other known genetic markers, such as the bcr-abl gene. Therefore, quantitative monitoring of the PRAME gene using real-time PCR method may be useful for detecting minimal residual disease and to predict subsequent relapse, especially in patients without known genetic markers. In addition, a PRAME-positive leukaemia cell line and fresh leukaemic cells were found to be susceptible to lysis by PRAME-specific CTLs established from a patient with melanoma, suggesting that the PRAME peptide can also be a target leukaemia antigen for T cells.

Bone morphogenetic protein-2 induces apoptosis in human myeloma cells with modulation of STAT3.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-beta superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2-induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21(CIP1/WAF1) and p27(KIP1)) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-x(L); however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-x(L) and by cell-cycle arrest through the up-regulation of p21(CIP1/WAF1) and p27(KIP1) and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7.

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.

Arsenic trioxide (As2O3)-induced apoptosis and differentiation in retinoic acid-resistant acute promyelocytic leukemia model in hGM-CSF-producing transgenic SCID mice.

Recent clinical studies in China and USA showed that arsenic trioxide (As2O3) is an effective treatment of acute promyelocytic leukemia (APL) patients refractory to all-trans retinoic acid (RA). We here investigate the effects of As2O3 on RA-resistant APL in vivo and in vitro using our RA-resistant APL model system. As2O3 can induce inhibition of cellular growth of both RA-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis in vitro. The expression of BCL-2 protein decreased in a dose- and time-dependent manner in NB4 cells. Interestingly, the levels of BCL-2 protein were not modulated by As2O3, but it did upregulate BAX protein in UF-1 cells. UF-1 cells (1x10(7)) were transplanted into hGM-CSF-producing transgenic SCID mice and successfully formed subcutaneous tumors. After 40 days of implantation, mice were treated with As2O3, all-trans RA and PBS for 21 days. In all-trans RA- and PBS-treated mice, tumors grew rapidly, with a 4.5-fold increase in volume at day 21 compared to the initial size. In marked contrast, tumor size was decreased to half of the initial size by the treatment of As2O3, which resulted in cells with the typical appearance of apoptosis. Interestingly, one of the As2O3-treated mice showed mature granulocytes in the diminished tumor, suggesting that As2O3 had dual effects on RA-resistant APL cells in vivo: both inducing apoptosis and differentiation of the leukemic cells. We conclude that our RA-resistant APL model will be useful for evaluating novel therapeutic approaches to patients with RA-resistant APL, and for further investigation of the metabolism of As2O3 in vivo.

Hypercalcemia after High-Dose Chemoradiotherapy for Refractory Multiple Myeloma; Subject Heading.

A 43-year-old man with refractory myeloma underwent allogeneic bone marrow transplantation from his HLA-matched sibling. He was conditioned with TBI (12 Gy) followed by melphalan (140 mg/m(2) ). Immediately after conditioning was initiated, he began complaining of severe lumbago, and the level of serum calcium rose from 2.25 to 3.34 mmol / l. However, the biochemical markers for tumor-lysis syndrome such as potassium, uric acid, and lactic dehydrogenase remained unchanged. Hydration with saline and pamidronate were started, but he developed acute renal failure requiring hemodialysis for 3 weeks. His plasma parathyroid hormonerelated protein (PTHrP)-NH2-terminal (3.9 pmol/l) and serum PTHrP-C-terminal (125.0 pmol / l) levels markedly increased immediately after conditioning. These results suggested that the increased release of PTHrP from myeloma cells, which resulted from destruction of myeloma cells by conditioning, was the primary contributes to the occurrence of hypercalcemia. We should be aware of the occurrence of hypercalcemia when high-dose therapy is to be given to patients with refractory myeloma.

A novel retinoic acid-resistant acute promyelocytic leukemia model in vitro and in vivo (review).

Differentiation-inducing therapy by all-trans retinoic acid (RA) is now a standard therapy in patients with acute promyelocytic leukemia (APL). Nearly all patients achieve complete remission by the treatment of all-trans RA, however, clinical remissions are usually of brief duration, and these patients often develop RA-resistant disease. The mechanisms of RA-resistance in APL cells are poorly understood and most clinical approaches have not been successful in overcoming RA-resistance. We have recently established a novel APL cell line (UF-1) with RA-resistant features. In addition, we have established human GM-CSF-producing transgenic (hGMTg) SCID mice system. UF-1 cells were inoculated either intraperitoneally or subcutaneously into hGMTg SCID mice and made the first RA-resistant murine APL model. These RA-resistant APL model systems in vitro and in vivo may be useful for investigating the molecular studies on the block of leukemic cell differentiation and as means to investigate the mechanisms of RA-resistance. Moreover, this murine model system will be important for developing novel therapeutic strategies in RA-resistant APL.

[Natural killer-like T-cell lymphoma appearing in the duodenum with recurrence in a variety of extranodal organs].

A 48-year-old man was admitted to Keio University Hospital in April 1995 with complaints of right abdominal pain and weight loss. Hypotonic duodenography showed a mass located in the 3rd portion of the duodenum. Endoscopic biopsy specimens disclosed diffuse large cell non-Hodgkin's lymphoma of the duodenum. The patient was initially treated with 2 courses of CHOP, albeit with no response. A pancreatoduodenectomy and radiotherapy yielded a complete remission. A year later, lymphoma recurred in the right mandible salivary gland, and a second complete remission was obtained after 6 courses of CHOP and radiation. However lymphomas also recurred in the intestine, and lungs, and the patient died of disease progression 38 months after diagnosis. Lymphoma cells were surface CD3 and CD56 positive. An examination of resected intestinal tissues disclosed lymphoma cells morphologically resemble large granular lymphocytes with rearranged TcR genes. These findings indicated the diagnosis of natural killer-like (NK-like) T-cell lymphoma. Compared with previously reported cases of NK-like T-cell lymphoma, this case was noteworthy for an unusual clinical course characterized by initial appearance in the duodenum, recurrence in a variety of extranodal organs, and the relatively long-term survival of the patient.

Human acute myeloblastic leukemia-ascites model using the human GM-CSF- and IL-3-releasing transgenic SCID mice.

To generate an appropriate model for human acute myeloblastic leukemia (AML), we have successfully established a human hematopoietic growth factor-dependent AML cell line (TF-1 and UT-7/GM)-ascites model using human granulocyte-macrophage colony-stimulating factor (hGM-CSF)- and human interleukin 3 (hIL-3)-releasing transgenic (Tg)-SCID mice. When 1 x 10(7) cells of TF-1, a human erythroleukemia cell line, were transplanted into the peritoneum of irradiated Tg-SCID mice (TF-1 ip/Tg-SCID mice), TF-1 cells grew in both the single cell suspension form (asTF-1) and solid form in ascites and invaded various tissues: lungs, liver, pancreas, and genitals, 3-6 weeks following transplantation. Subsequently, 0.5-1 x 10(7) cells of UT-7/GM, a subline of the UT-7 human megakaryoblastic leukemia cell line, grown in the back of hGM-CSF Tg-SCID mice after subcutaneous inoculation, were transplanted into the peritoneum of other irradiated hGM-CSF Tg-SCID mice. After 4 weeks, UT-7/GM cells (asUT-7/GM) also grew in the same manner as TF-1 cells in hGM-CSF Tg-SCID mice. Analysis of the cells from the peritoneum and tissues by PCR amplifying ALU and human GM-CSF receptor beta sequences and by immunohistochemical staining using anti-human CD45 revealed that they possessed the original characteristics of the parental cells. To confirm the usefulness of this human AML-ascites model, experimental treatment of AML cells grown in these mice was carried out with a differentiation inducer, delta-aminolevulinic acid (deltaALA), which induces hemoglobin synthesis for TF-1 in vitro and is thus regarded as an anti-leukemia drug candidate. Unexpectedly, growth promotion of TF-1 cells was observed in the treated TF-1 ip/hIL-3 Tg-SCID mice without differentiation to erythroid cells after treatment with delta-ALA (5 mM) for 7 days. These results indicate that Tg-SCID mice can support the growth of human hematopoietic growth factor-dependent AML cell lines which are usually rejected by SCID mice, without modification of the parental cell characteristics. In addition, this Tg-SCID leukemia-ascites model may become a useful preclinical tool for estimation of drug efficacy in vivo, since the drug candidate which was promising in vitro did not act in the same manner in vivo.