Investigation into Antiepileptic Effect of Ganoderic Acid A and Its Mechanism in Seizure Rats Induced by Pentylenetetrazole.

Ganoderic acid A (GAA) exhibited neuron protection in in vitro epilepsy study, but no study has been done in vivo. Rats were administered (i.p.) pentylenetetrazole daily for 28 days to induce seizure. Rats with grade II or above of epileptic score were divided into three groups and given placebo, sodium valproate, or GAA treatment, respectively, for 7 days. The electrical signals of brain were monitored with electroencephalography (EGG); epileptic behavior was assessed using the Racine scale; morphological changes and apoptosis rate of cortical neurons were assessed with H&E staining and TUNEL staining, respectively. Protein expression of calcium-sensing receptor, p-ERK, p-JNK, and p-p38 in hippocampal tissue and Bcl-2, cleaved caspase-3, and Bax in cortical tissues was observed by Western blot and immunohistochemistry assay, respectively. After GAA treatment, apparent seizure-like EEG with significant arrhythmic disorder and spike waves was reduced or disappeared, and wave amplitude of EEG was reduced significantly. GAA showed similar effect with sodium valproate treatments on epilepsy. There were an apparent improvement of the epileptic behavior and a significant increase in the epileptic latency and shortening of the epileptic duration in the treatment group compared to control. GAA treatment ameliorated the nuclear pyknosis of neurons which appeared seriously in the epilepsy group. GAA treatment significantly reduced the cortical neuron apoptosis of epilepsy and the expression of calcium-sensing receptor, p-P38, p-JNK, cleaved caspase-3, and Bax but increased the expression of both p-ERK and Bcl-2. In conclusion, GAA treatment showed strong antiepileptic effect by decreasing apoptosis in cortical neuron and the expression of calcium-sensing receptor and stimulating the MAPK pathway.

High expression of neuroguidin increases the sensitivity of acute myeloid leukemia cells to chemotherapeutic drugs.

Neuroguidin (NGDN) is a eukaryotic translation initiation factor 4E binding protein. The purpose of this study was to clarify the function of NGDN and its possible mechanism of action in human myeloid leukemia cells. Proliferation inhibition and apoptosis in NGDN over-expressing myeloid multidrug-resistant leukemia cells (K562/A02-NGDN) was significantly higher than in control K562/A02 cells following treatment with vincristine, etoposide, and epirubicin, indicating that NGDN over-expression can increase the sensitivity of multidrug-resistant leukemia cells to chemotherapeutic drugs. Furthermore, NGDN knock-down in K562/A02 cells resulted in the activation of multiple tumor-related signaling pathways, especially the mammalian target of rapamycin (mTOR) pathway.

High prognostic value of minimal residual disease detected by flow-cytometry-enhanced fluorescence in situ hybridization in core-binding factor acute myeloid leukemia (CBF-AML).

Acute myeloid leukemia (AML) is generally regarded as a disorder of stem cells, known as leukemic initiating cells (LICs), which initiate the disease and contribute to relapses. Although the phenotype of these cells remains unclear in most patients, they are enriched within the CD34(+)CD38(-) population. In core-binding factor (CBF) AML, the cytogenetic abnormalities also exist in LIC. The aim of this study was to determine the prognostic power of minimal residual disease (MRD) measured by fluorescence in situ hybridization (FISH) in CD34(+)CD38(-) cells sorted by flow cytometry at different periods during therapy. Thirty-six patients under 65 years of age with de novo CBF-AML treated with intensive chemotherapy were retrospectively included in this study. Correlations with relapse-free survival (RFS) and overall survival were evaluated with univariate and multivariate analyses. FISH efficiently identified LICs in the CD34(+)CD38(-) population. The presence of FISH(+)CD34(+)CD38(-) cells before consolidation was negatively associated with cumulative incidence of relapse (64 vs 18 %, P = .012), which showed prognostic value for RFS (12 vs 68 %, P = .008) and OS (11 vs 75 %, P = .0005), and retained prognostic significance for RFS in multivariate analysis. The detection of FISH(+)CD34(+)CD38(-) cells before consolidation therapy significantly correlated with long-term survival. Fluorescence-activated cell sorting (FACS)-FISH could be potentially adopted as a MRD monitor approach in clinical practice to identify CBF-AML patients at risk of treatment failure during therapy.

Characteristics of acute myeloid leukemia with myelodysplasia-related changes: A retrospective analysis in a cohort of Chinese patients.

We retrospectively analyzed 449 patients with AML under the WHO classification of AML 2008 and probed implications of this classification in diagnosis and treatment of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) among them. The clinical presentations, biological features, treatments, and prognosis of patients diagnosed with AML-MRC were analyzed and compared with those of AML not otherwise specified (AML-NOS). In all patients, 115 (25.6%) were diagnosed as AML-MRC including 64 males and 51 females with median onset age of 48 years (range from 17 to 78). Their complete remission (CR) rate was 60.9% and relapse rate was 57.1%. The observed median overall survival (OS) and disease-free survival (DFS) were 10 and 5 months, respectively, which was significantly shorter than those of AML-NOS patients (P < 0.05). The prognosis of AML-MRC patients with myelodysplastic syndrome (MDS)-related cytogenetics sole was similar to those with history of MDS or myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Patients with MDS-related cytogenetic abnormalities and/or history of MDS or MDS/MPN predisposed significantly shortened CR, OS, and DFS than AML-MRC patients with only multilineage dysplasia (MLD) and AML-NOS patients (P < 0.05). Multivariate analysis showed that age, cytogenetics, and history of MDS or MDS/MPN were independent prognostic factors. Patient diagnosed as AML-MRC presented distinctive clinical and biological features. Presence of MLD does not change the prognosis.

Incorporation of arsenic trioxide in induction therapy improves survival of patients with newly diagnosed acute promyelocytic leukaemia.

OBJECTIVES: For patients with acute promyelocytic leukaemia (APL), negative reading of a promyelocytic leukaemia/retinoic acid receptor-alpha (PML-RARα) transcript after induction therapy correlates with a good prognosis. However, in the majority of patients given all-trans retinoic acid (ATRA)/anthracycline-based induction therapy, PML-RARα transcript remains even when haematologic complete remission is achieved. To facilitate maximal therapeutic efficacy for patients with APL, this study tested whether the addition of arsenic trioxide (ATO) would increase the rate of molecular complete remission after ATRA/anthracycline-based induction therapy. METHODS: Seventy-three patients with APL were induced with a regimen (designated 'AAA') consisting of ATO in combination with ATRA and daunorubicin. After this, a consolidation phase of daunorubicin-based chemotherapy and maintenance therapy with ATRA, ATO and methotrexate was administered. The noted outcomes were rates of complete remission, overall survival and disease-free survival. In addition, PML-RARα transcripts were monitored in 48 patients via RT-PCR. RESULTS: Rates of complete remission, overall survival and 5-yr disease-free survival were 95.89%, 94.52% and 96.28%, respectively. At the preconsolidation checkpoint, 68.75% (33/48) of patients had a negative reading for the PML-RARα fusion transcript. These outcomes were not influenced by mutations in FLT3 (fms-related tyrosine kinase 3) or other prognostic factors. CONCLUSIONS: The addition of ATO in the induction regimen was associated with an improved overall outcome for patients with de novo APL, with rather low relapse rate and better long-term survival.

Homoharringtonine and omacetaxine for myeloid hematological malignancies.

Homoharringtonine (HHT), a plant alkaloid with antitumor properties originally identified nearly 40 years ago, has a unique mechanism of action by preventing the initial elongation step of protein synthesis. HHT has been used widely in China for the treatment of chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Omacetaxine, a semisynthetic form of HHT, with excellent bioavailability by the subcutaneous route, has recently been approved by FDA of the United States for the treatment of CML refractory to tyrosine kinase inhibitors. This review summarized preclinical and clinical development of HHT and omacetaxine for myeloid hematological malignancies.

The resistance mechanisms of proteasome inhibitor bortezomib.

The proteasome inhibitor, bortezomib, a boronic dipeptide which reversibly inhibit the chymotrypsin-like activity at the ß5-subunit of proteasome (PSMB5), has marked efficacy against multiple myeloma and several non-Hodgkin's lymphoma subtypes, and has a potential therapeutic role against other malignancy diseases. However, intrinsic and acquired resistance to bortezomib may limit its efficacy. In this article, we discuss recent advances in the molecular understanding of bortezomib resistance. Resistance mechanisms discussed include mutations of PSMB5 and the up-regulation of proteasome subunits, alterations of gene and protein expression in stress response, cell survival and antiapoptotic pathways, and multidrug resistance.

FISH+CD34+CD38- cells detected in newly diagnosed acute myeloid leukemia patients can predict the clinical outcome.

BACKGROUND: In acute myeloid leukemia (AML), the leukemia initiating cells (LICs) or leukemia stem cells (LSCs) is found within the CD34+CD38- cell compartment. The LICs subpopulation survives chemotherapy and is most probable the cause of minimal residual disease (MRD), which in turn is thought to cause relapse. The aim of this study was to determine the prognostic value of the percentage of LICs in blasts at diagnosis. DESIGN AND METHODS: The percentage of LICs in the blast population was determined at diagnosis using a unique Flow-FISH analysis, which applies fluorescent in situ hybridization (FISH) analysis on flow cytometry sorted cells to distinguish LICs within the CD34+CD38- cell compartment. Fourty-five AML patients with FISH-detectable cytogenetic abnormalities treated with standardized treatment program were retrospectively included in the study. Correlations with overall survival (OS), events-free survival (EFS) and cumulative incidence of relapse (CIR) were evaluated with univariate and multivariate analysis. RESULTS: The percentage of LICs is highly variable in patients with acute myeloid leukemia, ranged from 0.01% to 52.8% (median, 2.1%). High LIC load (≥1%) negatively affected overall survival (2-year OS: 72.57% vs. 16.75%; P = 0.0037) and events-free survival (2-year EFS: 67.23% vs. 16.33%; P = 0.0018), which was due to an increased cumulative incidence of relapse (2-year CIR: 56.7% vs. 18.0%; P = 0.021). By multivariate analysis, high LIC load retained prognostic significance for OS and EFS. CONCLUSIONS: In the present study, we established the Flow-FISH protocol as a useful method to distinguish normal and leukemic cells within the CD34+CD38- cell subpopulation. The high percentage of LICs at diagnosis was significantly correlated with increased risk of poor clinical outcome.

Bortezomib suppresses the growth of leukemia cells with Notch1 overexpression in vivo and in vitro.

Bortezomib has been widely used in the treatment of various cancers; however, its exact mechanisms of action are not fully understood, particularly in acute T lymphoblast leukemia (T-ALL). Here, we visualize the anti-leukemia effect of bortezomib in both human T-ALL cell line and animal models. In vitro study, a human T-ALL cell line bearing Notch1 mutations, MOLT-4, was treated with bortezomib. At clinically achievable concentrations, bortezomib inhibited cell growth by inducing G1 phase arrest and apoptosis with a dose-dependent manner. A murine tumor xenograft model was achieved by subcutaneous injection of MOLT-4 cells for in vivo study. Administration of bortezomib significantly reduced tumor mass volume when compared with controls. Of note, bortezomib inhibited growth of leukemia cells in a Notch1-induced murine T-ALL model, and the life span of leukemia-bearing mice was markedly increased. Further studies revealed that bortezomib led to inhibited expression of Notch1 target genes. Taken together, our results demonstrate that bortezomib shows significant anti-leukemia effect in T-ALL bearing Notch1 mutations in vitro and in vivo. The present study provides evidence that bortezomib might be a candidate therapeutic reagent in the treatment of T-ALL.

[Comparison of protein expression profiles between bortezomib-resistant JurkatB cells with PSMB5 mutation and their parent cells].

This study was purposed to investigate the differences of cyto biological characteristics and protein expression levels between bortezomib-resistant T-lymphoblastic lymphoma/leukemia cell lines JurkatB containing PSMB5 G322A mutation and their parent cell line Jurkat, The cytotoxicities of bortezomib and chemotherapeutic drugs to JurkatB5 cells (end selection concentration of bortezomib was 500 nmol/L), JurkatB8 (end selection concentration 800 nmol/L) and Jurkat cells were analyzed. The cell growth curves were drawn with viable cell counts by trypan blue assay, the colony formation rate were assayed by soft-agar colony culture, and the cell distributions in cell cycle were analyzed by flow cytometry, mRNA expression levels of multidrug resistance (MDR) genes MDR1, LRP and MRP were measured by real-time fluorescence quantitative RT-PCR, the differences of protein expression levels were detected by SpringBio antibody microarray containing 720 proteins. The results showed that the drug resistance multiples for 48 hours of JurkatB5 and JurkatB8 cells (relative to Jurkat) to bortezomib were increased by 33.52 and 39.04 times, respectively. JurkatB5 and JurkatB8 cells did not display significant cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide after exposure for 48 hours. There were no significant differences in the cell growth curve, colony formation rate and cell distributions in cell cycle between JurkatB5, JurkatB8 and Jurkat cells (p > 0.05). There were no significant differences of mRNA expression levels of MDR1, LRP, MRP between JurkatB5 and Jurkat cells (p > 0.05). There were 264 analyzable expression points detected by antibody microarray. Among them, 252 protein expression levels were not significantly different between JurkatB5, JurkatB8 and Jurkat cells (< 2-fold), including 15 drug resistance-related proteins. 12 proteins were detected at higher or lower expression levels in JurkatB5 or JurkatB8 cells then that in Jurkat cells (cell division cycle protein 34, cell division cycle protein 37, CD34 Type II, matrix metalloproteinase-2, tenascin, Golgi complex, involucrin, histone deacetylase 1, perforin, prolactin, retinoic acid receptor ß, integrin ß-1), but no proteins were detected in JurkatB5 and JurkatB8 cells with higher or lower expression levels than that in Jurkat cells. It is concluded that there are no significant differences in the characteristics of cellular biology between Jurkat and JurkatB with bortezomib-resistant and PSMB5 G322A mutation. There are no significant phenotype change of MDR and overexpression of genes related to MDR in PSMB5 mutated cells. There are no significantly differential expressions of a majority of known proteins related to drug resistance, tumor cells growth, proliferation, apoptosis, malignancy degree, aggressiveness.

A homoharringtonine-based induction regimen for the treatment of elderly patients with acute myeloid leukemia: a single center experience from China.

BACKGROUND AND PURPOSE: The response to remission induction in elderly patients with acute myeloid leukemia (AML) remains poor. The purpose of this paper is to evaluate the efficacy and toxicity of a plant alkaloid, homoharringtonine, in combination with cytarabine as an induction therapy for AML in elderly patients (> or =60 years). RESULTS: Twenty-three patients were treated with the HA regimen consisting of homoharringtonine (2 mg/m2/day for 7 days) and cytarabine (Ara-C, 100 mg/m2/day for 7 days). The overall response rate was 56.5% with complete remission (CR) rate of 39.1% and partial remission of 17.4%. There was no early death in this cohort of patients. The estimated median overall survival (OS) time of all patients was (12.0 +/- 3.0) months. The estimated OS time of the CR patients was 15 months. The estimated one-year OS rate of all patients treated with HA protocol was (49.3 +/- 13.5) %. The estimated one-year OS rate of the CR patients was (62.5 +/- 17.1) %. CONCLUSION: HA is a suitable induction regimen for elderly patients with AML, with relatively low toxicity and reasonable response rate.

Clinical and biological characteristics of adult biphenotypic acute leukemia in comparison with that of acute myeloid leukemia and acute lymphoblastic leukemia: a case series of a Chinese population.

BACKGROUND: Biphenotypic acute leukemia is a rare disorder that is difficult to diagnose. It displays features of both myeloid and lymphoid lineage. There is still a lack of studies in biphenotypic acute leukemia in a Chinese population. We present here a comprehensive investigation of the clinical and biological characteristics, and outcome of biphenotypic acute leukemia in our hospital in over a seven year period. DESIGN AND METHODS: We retrospectively analyzed 452 adult acute leukemia patients diagnosed according to French-American-British (FAB) classification and biphenotypic acute leukemia diagnosed according to European Group for the Immunological Characterization of Leukemias (EGIL) classification, respectively. Biological characteristics, response to treatment, and outcome were examined in biphenotypic acute leukemia patients and compared with that in acute myeloid leukemia and acute lymphoblastic leukemia patients with complete follow-up profiles diagnosed in the same period. RESULTS: Of 452 acute leukemia patients, 21 cases (4.6%) were diagnosed as biphenotypic acute leukemia. Among them, 14 (66.7%) were B lymphoid and myeloid, 5 (23.8%) were T lymphoid and myeloid, one (4.8%) was T/B lymphoid and one (4.8%) was trilineage differentiation. When compared with acute myeloid leukemia and acute lymphoblastic leukemia, patients with biphenotypic acute leukemia showed significantly higher incidence of CD34 antigen expression, unfavorable karyotypes, and extramedullary infiltration (p<0.05). In this cohort of patients with biphenotypic acute leukemia, t(9;22) was the most common abnormality in chromosome structure. The median disease-free survival and overall survival in biphenotypic acute leukemia patients was five months and ten months, respectively, significantly shorter than those in acute myeloid leukemia and acute lymphoblastic leukemia patients (p<0.05). CONCLUSIONS: The prognosis of biphenotypic acute leukemia patients is poor when compared with de novo acute myeloid leukemia or acute lymphoblastic leukemia. Biphenotypic acute leukemia patients showed a much higher incidence of CD34 antigen expression, complex abnormal karyotype, extramedullary infiltration, relapse, and resistance to therapy after relapse.

Different mutants of PSMB5 confer varying bortezomib resistance in T lymphoblastic lymphoma/leukemia cells derived from the Jurkat cell line.

OBJECTIVE: To investigate the relationship between bortezomib resistance and mutations in the proteasome beta5 subunit (PSMB5) gene. MATERIALS AND METHODS: Various bortezomib-resistant lymphoblastic lymphoma/leukemia lines were established by repeated cycles of bortezomib selection. Mutations were detected by sequencing the complementary DNA of the PSMB5 gene. Mutated clones were selected by limited dilution and cultured without bortezomib. Messenger RNA expression levels of PSMB5 in these mutated clones were measured by quantitative reverse transcription polymerase chain reaction. The degree of resistance was determined by cytotoxicity at various bortezomib concentrations. The chymotrypsin-like activities were assayed by measuring the release of the fluorescent 7-amido-4-methylcoumarin from the substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. RESULTS: In addition to the previously reported PSMB5 G322A mutant (Ala49Thr), a C323T mutant (Ala49Val), and G322A, C326T conjoined mutant (Ala49Thr and Ala50Val) were selected and clones containing these mutations (JurkatB-G322A, JurkatB-C323T, and JurkatB-G322A/C326T) were obtained. After being cultured without bortezomib for >2 months, no significant difference in PSMB5 messenger RNA levels was detected between these JurkatB cells and parental Jurkat cells. JurkatB-G322A, JurkatB-C323T, and JurkatB-G322A/C326T clones displayed 22.0-fold, 39.4-fold, and 66.7-fold resistance, respectively, to bortezomib compared to Jurkat cells. There were no significant differences between the chymotrypsin-like activities of these mutants and Jurkat cells. The inhibitory effect of bortezomib on chymotrypsin-like activity was the weakest in JurkatB-G322A/C326T cells, and the strongest in JurkatB-G322A cells, with JurkatB-C323T cells falling in between. CONCLUSION: Mutations of the PSMB5 gene resulting in substitutions of Ala49 and Ala50 of PSMB5 protein can confer varying bortezomib resistance.

Bortezomib in combination with epirubicin, dexamethasone and thalidomide is a highly effective regimen in the treatment of multiple myeloma: a single-center experience.

The aim of the present study was to evaluate the effectiveness of bortezomib combined with epirubicin, dexamethasone, and thalidomide (BADT) for the treatment of multiple myeloma (MM). The BADT regimen consisted of a maximum of eight 4-week cycles of: intravenous bortezomib (1.0 mg/m(2)) and intravenous epirubicin (12 mg/m(2)) on days 1, 4, 8, and 11; dexamethasone (20 mg) on days 1, 2, 4, 5, 8, 9, 11, and 12; and oral thalidomide (100 mg/m(2)) on days 1-28. Twelve patients with MM were included in the study, of whom four had not been previously treated and eight had been previously treated with at least one cycle of a systemic combined regimen. All the patients completed at least two cycles of treatment, with an average of five cycles; the complete response (CR) rate was 83.3% (10/12) and stabilization of disease was 16.7% (2/12). The average number of cycles required to achieve CR was 1.9 (range 1-6). In three patients, mobilization of peripheral blood stem cells allowed a sufficient quantity of CD34+ cells to be harvested for future autotransplantation. The main adverse reactions included peripheral neuropathy (4/12), thrombocytopenia (3/12), electrocardiographic abnormalities (4/12), neutropenia (5/12), and liver function impairment (4/12), primarily grade I-II. Infection occurred in four patients with neutropenia, including one patient who developed sepsis. The estimated 1-year overall survival rate was 91.7 +/- 8.0%, and the estimated 1-year disease-free survival was 75.0 +/- 12.5%. BADT is a highly effective combined regimen, with acceptable toxicity, for the treatment of multiple myeloma.

[Synergistic effects of arsenic trioxide and proteasome inhibitor bortezomib on apoptosis induction in Raji cell line].

The aim of this study was to explore the synergistic effect of arsenic trioxide and bortezomib on apoptosis of Raji cell line. The cells were treated with arsenic trioxide, bortezomib, low-dose arsenic trioxide combined with bortezomib, respectively. The cell viability and proliferative curve were estimated by trypan blue dye exclusion. The cell apoptosis and cell cycle status were analyzed by flow cytometry. The apoptosis related elements such as caspase-3, BCL-2, BAX, JNK2 and IkappaB-alpha, were measured with Western blot. The results showed that compared with cells treated with mentioned above drugs alone, the proliferative potential of cells in combination group was significantly inhibited (p < 0.01), and apoptosis rate markedly increased (p = 0.001), while obvious cell cycle arrest was not observed. On the protein level, the expression of Caspase-3, BAX and IkappaB-alpha increased, while the expression of BCL-2, and JNK2 decreased. It is concluded that low-dose arsenic trioxide combined with bortezomib synergistically induced apoptosis in Raji cell line which may be mediated by inhibiting NK-kappaB and JNK2 signaling.

Overexpression of the PSMB5 gene contributes to bortezomib resistance in T-lymphoblastic lymphoma/leukemia cells derived from Jurkat line.

OBJECTIVE: To study the mechanism of bortezomib resistance in JurkatB lines derived from T-lymphoblastic lymphoma/leukemia Jurkat line. MATERIALS AND METHODS: Cytotoxicities of popular chemotherapeutic drugs to JurkatB cells were analyzed by trypan blue assay. Functional drug efflux in JurkatB cells was determined by flow cytometry utilizing daunorubicin and the expression of P-glycoprotein (P-gp) was detected by Western blot. mRNA expression levels of proteasome beta5 subunit (PSMB5) were measured by quantitation real-time reverse transcription polymerase chain reaction. In situ hybridization was performed to detect the amplification of PSMB5 gene. The chymotrypsin-like activities were assayed by measuring the release of the fluorescent 7-amido-4-methylcoumarin (AMC) from the substrate N-succinyl-Leu-Leu-Val-Tyr-AMC. Cytogenetic studies were performed using R-banded metaphases and fluorescence in situ hybridization (FISH) analysis. IkappaB-alpha levels were detected by Western blot. RESULTS: No cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide was found in JurkatB cells. No evidence of drug efflux was found in JurkatB cells and the expression of P-gp was negative. The PSMB5 mRNA was overexpressed in highly resistant JurkatB5 and JurkatB1 lines compared with parental Jurkat, corresponding well with the increase of chymotrypsin-like activity and a karyotype of i(14q). Amplification of PSMB5 gene was demonstrated by in situ hybridization and FISH. The decreased IkappaB-alpha level in JurkatB5 cells after bortezomib treatment indicating an upregulation of nuclear factor-kappaB (NF-kappaB) activity. CONCLUSION: The mechanism of bortezomib resistance is different from that of multidrug resistance. Overexpression of PSMB5 is an important mechanism for bortezomib resistance in JurkatB lines. NF-kappaB may play a critical role in evading the apoptotic effects.

Point mutation of the proteasome beta5 subunit gene is an important mechanism of bortezomib resistance in bortezomib-selected variants of Jurkat T cell lymphoblastic lymphoma/leukemia line.

To study the mechanism of acquired resistance to bortezomib, a new antitumor drug that is the first therapeutic proteasome inhibitor, we established a series of bortezomib-resistant T lymphoblastic lymphoma/leukemia cell lines, designated the JurkatBs, from the parental Jurkat line via repeated drug selection. There were no significant differences in the growth curves or colony formation between the JurkatB cells and parental Jurkat cells. The effects of bortezomib on cytotoxicity, cell cycle arrest, and induction of apoptosis were decreased in JurkatB cells compared with parental Jurkat cells. A mutation in the proteasome beta5 subunit (PSMB5) gene (G322A), which encodes an amino acid change from Ala to Thr at polypeptide position 108, was detected by sequencing full-length cDNA clones and direct polymerase chain reaction products of the PSMB5 gene. Bortezomib caused less inhibition of chymotrypsin-like activity in resistant cells. When the G322A mutant PSMB5 was retrovirally introduced into parental Jurkat cells, it conferred bortezomib resistance to these cells, resulting in decreased cytotoxicity, apoptosis, and inhibition of chymotrypsin-like activity. The predicted structure of A108T-mutated PSMB5 shows a conformational change that suggests decreased affinity to bortezomib. In short, the G322A mutation of the PSMB5 gene is a novel mechanism for bortezomib resistance.

[An analysis of prognostic factors in 80 myelodysplastic syndrome].

OBJECTIVE: To investigate the WHO classification, clinical and hematological features and risk group of International Prognostic Scoring System (IPSS) in patients with myelodysplastic syndrome (MDS). METHODS: The diagnosis and classification of MDS patients were defined according to the WHO classification. The clinical manifestations, hemogram, bone marrow biopsy and prognosis were retrospectively analyzed. RESULTS: The median age at diagnosis of MDS was 47 yrs being younger than that in some foreign reports. The frequency of abnormal karyotype was 35.14% and +8 was the most frequent abnormal karyotype in our study. Eleven of 74 patients transformed into leukemia. Univariate analysis showed that age, chromosome abnormality, percentage of bone marrow blast cells and number of cytopenias were significantly related to prognosis. There was a statistical difference in cum survival rate between IPSS subcategories (P < 0.05) except that between low- and intermediate I-risk subcategory (P > 0.05). There were statistical differences for refractory anemia (RA) vs RA with excess blast (RAEB), refractory cytopenias with multilineage dysplasia (RCMD) vs RAEB and RAEB-I vs RAEB-II (P < 0.05). CONCLUSIONS: There were differences in age of disease onset, distribution of WHO, sub-classification and abnormal karyotype in this cohort of MDS patients as compared with those in Europe and Japan. It is helpful in diagnosis, treatment and prognosis to divide RAEB into RAEB-I and RAEB-II. IPSS was well applicable in Chinese MDS patients.

[Effects of proteasome inhibitors on leukemias].

The proteasome is primarily responsible for intracellular protein degradation. The abnormality of its activity is sign of tumorigenesis. It was confirmed that proteasome inhibitors have activities against a variety of malignancies. Bortezomib, the first proteasome inhibitor, obtained permission of clinical trial and on sale. Multiple myeloma patients treated with bortezomib have gained a high overall response rate and complete remission rate. A lot of studies on effects of proteasome inhibitors on leukemias, including plasma cell leukemia; chronic lymphocytic leukemia, adult T cell lymphoma/leukemia, chronic myeloid leukemia and acute myeloid leukemia, were reviewed in this article.

[A clinical observation of fludarabine-containing regimens in the treatment of low grade non-Hodgkin's lymphoma].

OBJECTIVE: To evaluate the therapeutic efficiency and adverse effect of the fludarabine-containing regimens in the treatment of low grade non-Hodgkin's lymphoma. METHODS: Thirty-two patients with low grade non-Hodgkin's lymphoma consisting of 19 primary one and 13 relapsed or refractory were treated with fludarabine-containing regimens, which included FMD (fludarabine, mitoxantrone and dexamethasone); FMC (fludarabine, cyclophosphamide and mitoxantrone) and FC ( fludarabine and cyclophosphamide). RESULTS: The average course completed in these 32 patients was 4.1 with a complete response rate (CR), partial response rate (PR) and overall response rate (OR) of 65.6%, 18.8% and 84.4% , respectively. There were no significant difference in CR, PR and OR between primary and relapsed or refractory group (71.4%, 21.0%, 92.4% vs. 46.2%, 13.1%, 59.3%, respectively). Myelotoxicity and immunotoxicity was the dominating adverse effects. Ill to IV grade granulocytopenia and thrombocytopenia were observed in 31.3% (10/32) and 9.4% (3/32) of these patients respectively. Infection developed in 7 patients, and two of them died of pulmonary infection. The median follow-up period was 16 months (1-30 months) with 2-year overall-survival rate (OS) and progression-free survival rate (PFS) of 93.8% and 84.4%, respectively. No significant difference was observed between primary and relapsed or refractory group in OS (100% vs. 76.9%) and PFS (94.7% vs. 69.2%). CONCLUSION: Fludarabine-containing regimens is well tolerated and effective in the treatment of low grade non-Hodgkin's lymphoma.