Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL.

Acute promyelocytic leukaemia (APL), associated with chromosomal translocations involving the retinoic acid receptor alpha gene (RARA) and the PML gene, is sensitive to retinoic acid (RA) treatment, while APL patients harbouring translocations between RARA and the PLZF gene do not respond to RA. We have generated PML-RARA and PLZF-RARA transgenic mice and show here that these fusion proteins play a critical role in leukaemogenesis and in determining responses to RA in APL, because PLZF-RARA transgenic mice develop RA-resistant leukaemia, while PML-RARA mice are responsive to RA treatment. We demonstrate that both PML-RARalpha and PLZF-RARalpha fusion proteins can act as transcriptional repressors and are able to interact with nuclear receptor transcriptional co-repressors, such as SMRT. PLZF-RARalpha, but not PML-RARalpha, can form, via its PLZF moiety, co-repressor complexes which are insensitive to RA. Histone deacetylase inhibitors such as Trichostatin A (TSA), in combination with RA, can overcome the transcriptional repressor activity of PML-RARalpha and PLZF-RARalpha as well as the unresponsiveness of PLZF-RARalpha-expressing leukaemic cells to RA. Thus, our findings unravel a crucial role for transcriptional silencing in APL pathogenesis and resistance to RA in APL.

Role of promyelocytic leukemia (PML) protein in tumor suppression.

The promyelocytic leukemia (PML) gene encodes a putative tumor suppressor gene involved in the control of apoptosis, which is fused to the retinoic acid receptor alpha (RARalpha) gene in the vast majority of acute promyelocytic leukemia (APL) patients as a consequence of chromosomal translocations. The PMLRARalpha oncoprotein is thought to antagonize the function of PML through its ability to heterodimerize with and delocalize PML from the nuclear body. In APL, this may be facilitated by the reduction to heterozygosity of the normal PML allele. To determine whether PML acts as a tumor suppressor in vivo and what the consequences of deregulated programmed cell death in leukemia and epithelial cancer pathogenesis are, we crossed PML(-/-) mice with human cathepsin G (hCG)-PMLRARalpha or mammary tumor virus (MMTV)/neu transgenic mice (TM), models of leukemia and breast cancer, respectively. The progressive reduction of the dose of PML resulted in a dramatic increase in the incidence of leukemia, and in an acceleration of leukemia onset in PMLRARalpha TM. By contrast, PML inactivation did not affect neu-induced tumorigenesis. In hemopoietic cells from PMLRARalpha TM, PML inactivation resulted in impaired response to differentiating agents such as RA and vitamin D3 as well as in a marked survival advantage upon proapoptotic stimuli. These results demonstrate that: (a) PML acts in vivo as a tumor suppressor by rendering the cells resistant to proapoptotic and differentiating stimuli; (b) PML haploinsufficiency and the functional impairment of PML by PMLRARalpha are critical events in APL pathogenesis; and (c) aberrant control of programmed cell death plays a differential role in solid tumor and leukemia pathogenesis.

Histone deacetylase inhibitors induce remission in transgenic models of therapy-resistant acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations, invariably involving the retinoic acid receptor alpha (RAR alpha) gene fused to one of several distinct loci, including the PML or PLZF genes, involved in t(15;17) or t(11;17), respectively. Patients with t(15;17) APL respond well to retinoic acid (RA) and other treatments, whereas those with t(11;17) APL do not. The PML-RAR alpha and PLZF-RAR alpha fusion oncoproteins function as aberrant transcriptional repressors, in part by recruiting nuclear receptor-transcriptional corepressors and histone deacetylases (HDACs). Transgenic mice harboring the RAR alpha fusion genes develop forms of leukemia that faithfully recapitulate both the clinical features and the response to RA observed in humans with the corresponding translocations. Here, we investigated the effects of HDAC inhibitors (HDACIs) in vitro and in these animal models. In cells from PLZF-RAR alpha/RAR alpha-PLZF transgenic mice and cells harboring t(15;17), HDACIs induced apoptosis and dramatic growth inhibition, effects that could be potentiated by RA. HDACIs also increased RA-induced differentiation. HDACIs, but not RA, induced accumulation of acetylated histones. Using microarray analysis, we identified genes induced by RA, HDACIs, or both together. In combination with RA, all HDACIs tested overcame the transcriptional repression exerted by the RAR alpha fusion oncoproteins. In vivo, HDACIs induced accumulation of acetylated histones in target organs. Strikingly, this combination of agents induced leukemia remission and prolonged survival, without apparent toxic side effects.

Therapeutic targeting of transcription in acute promyelocytic leukemia by use of an inhibitor of histone deacetylase.

BACKGROUND: Acetylation of DNA-associated histones is linked to activation of gene transcription, whereas histone deacetylation is associated with transcriptional repression. Recent studies have shown that inhibitors of histone deacetylases can relieve transcriptional repression caused by the products of certain oncogenes. We tested whether these findings could be applied clinically to a patient with highly resistant acute promyelocytic leukemia. METHODS: A patient who had experienced multiple relapses was treated with all-trans-retinoic acid alone and in combination with sodium phenylbutyrate, an inhibitor of histone deacetylases. Immunohistochemistry and western blot analysis were used to assay for histone hyperacetylation in mononuclear cells from the patient's blood and bone marrow. Marrow mononuclear cells and reverse transcription-polymerase chain reaction (RT-PCR) analysis of messenger RNA encoded by the PML/RAR-alpha oncogene were used to assess minimal residual disease. RESULTS: The patient proved clinically resistant to treatment with all-trans-retinoic acid alone. However, 23 days after sodium phenylbutyrate was added to the treatment regimen, visible leukemic cells had been eliminated from her bone marrow, and she achieved a complete clinical and cytogenetic remission shortly thereafter. With a second treatment course, analysis for minimal residual disease by RT-PCR proved negative. Immunofluorescence and western blot analysis showed that phenylbutyrate caused a time-dependent increase in histone acetylation in blood and bone marrow mononuclear cells. CONCLUSIONS: Clinical treatment with an inhibitor of histone deacetylase induces histone hyperacetylation in target cells and may restore sensitivity to the anti-leukemic effects of all-trans-retinoic acid in acute promyelocytic leukemia. Similar therapy may prove useful in other neoplastic diseases that are associated with oncogenic repression of gene transcription due to recruitment of histone deacetylases.

Role of double-stranded RNA-activated protein kinase in human hematological malignancies.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is one of many genes induced by IFN. The PKR sequentially undergoes autophosphorylation and activation on binding to dsRNA. Previous studies have shown that PKR may be an important factor in the regulation of viral and cellular protein synthesis. Recent studies suggest that PKR may function as a tumor suppressor gene. The role of PKR in various human leukemic cells was therefore investigated. PKR mRNA levels by reverse transcription-PCR, protein expression by Western blot and FACScan analysis, and activity by phosphorylation status were studied. The expression of a known inhibitor of PKR, p58, was also investigated at mRNA and protein levels. A total of 24 samples from normal mononuclear cells (MNCs), 26 samples of acute lymphoblastic leukemia, 26 samples of acute myelogenous leukemia, 32 samples of chronic lymphocytic leukemia, and 5 samples of hairy cell leukemia was investigated. Mean mRNA levels were increased in acute lymphoblastic leukemia and acute myelogenous leukemia and decreased in chronic lymphocytic leukemia compared to normal MNCs. The mRNA levels in hairy cell leukemia were similar to those of normal MNCs. PKR protein was detectable in normal MNCs and leukemic cell extracts, and on FACScan analysis, more than 70% of cells stained positive for PKR. PKR activity was detectable in all samples investigated and was enhanced 4-23-fold in the presence of the synthetic dsRNA, poly(I) x poly(C). Protein expression of a known PKR inhibitor, p58, was barely detectable in normal MNCs and leukemic cells, with high expression in the HeLa cell line. These findings provide no evidence to support the hypothesis that PKR acts as a tumor suppressor in human leukemic cells.

In vivo analysis of the molecular pathogenesis of acute promyelocytic leukemia in the mouse and its therapeutic implications.

Acute promyelocytic leukemia (APL) is characterized by the expansion of malignant myeloid cells blocked at the promyelocytic stage of hemopoietic development, and is associated with reciprocal chromosomal translocations always involving the retinoic acid receptor alpha (RARalpha) gene on chromosome 17. As a consequence of the translocation RARalpha variably fuses to the PML, PLZF, NPM and NUMA genes (X genes), leading to the generation of RARalpha-X and X-RARalpha fusion genes. The aberrant chimeric proteins encoded by these genes may exert a crucial role in leukemogenesis. Retinoic acid (RA), a metabolite of vitamin A, can overcome the block of maturation at the promyelocytic stage and induce the malignant cells to terminally mature into granulocytes resulting in complete albeit transient disease remission. APL has become, for this reason, the paradigm for 'cancer differentiation therapy'. Furthermore, APL associated with translocation between the RARalpha and the PLZF genes (PLZF-RARalpha) shows a distinctly worse prognosis with poor response to chemotherapy and little or no response to treatment with RA, thus defining a new APL syndrome. Here we will focus our attention on the recent progresses made in defining the molecular mechanisms underlying the pathogenesis of this paradigmatic disease in vivo in the mouse. We will review the critical contribution of mouse modeling in unraveling the transcriptional basis for the differential response to RA in APL. We will also discuss how this new understanding has allowed to propose, develop and test in these murine leukemia models as well as in human APL patients novel therapeutic strategies.

RAS gene mutations in Chinese leukaemia patients and members of a family with high incidence of leukaemia.

We investigated mutations of N-RAS and K-RAS by using polymerase chain reaction (PCR)-oligonucleotide hybridization techniques in 40 cases of Chinese leukaemia patients and 17 presently healthy members of a family with high incidence of acute myeloid leukaemia. The results showed only two patients carried the mutation in codon 12 of N-RAS. Strikingly, however, in both cases the malignancies involved lymphoid lineage. There was no hereditary RAS mutation in the members of the remarkable family.

Genetic mechanism of leukemia predisposition in a family with 7 cases of acute myeloid leukemia.

This paper describes a family in which seven members, involving three consecutive generations, suffered from acute myeloid leukemia during the past 16 years. The genetic abnormalities were vertically transmitted in a Mendelian dominant manner without sex linkage. Cytogenetic approaches, such as G-banding karyotypes, micronuclei (MN), chromosome aberrations (CA), fragile sites (Fra), cell cycle time (Tc), sister chromatid exchanges (SCE), silver-staining nucleolar organizer regions (Ag-NOR), and silver-staining acrocentric chromosome satellite association (Ag-AA), were investigated on 19 presently healthy members of the family, compared with 10 normal controls. The results showed that their G-banding chromosome karyotypes were normal, without a single similarly located fragile site being found to be carried commonly by the blood relations, although the rare fragile site frequency in the blood relations group was higher than that in the non-blood relations group or normal controls. On the other hand, SCE and Ag-NOR were lowered in the blood relations. In III-13 and IV-3 of the pedigree, in particular, the prolonged cell cycle time with distinctly abnormal SCE and Ag-NOR might predict a high risk for leukemia. Hence the follow-up of this remarkable family is being continued.

Modeling and analysis of the affinity filtration process, including broth feeding, washing, and elution steps.

Affinity filtration is a developing protein purification technique that combines the high selectivity of affinity chromatography and the high processing speed of membrane filtration. In this work a lumped kinetic model was developed to describe the whole affinity filtration process, including broth feeding, contaminant washing, and elution steps. Affinity filtration experiments were conducted to evaluate the model using bovine serum albumin as a model protein and a highly substituted Blue Sepharose as an affinity adsorbent. The model with nonadjustable parameters agreed fairly to the experimental results. Thus, the performance of the affinity filtration in processing a crude broth containing contaminant proteins was analyzed by computer simulations using the lumped model. The simulation results show that there is an optimal protein loading for obtaining the maximum recovery yield of the desired protein with a constant purity at each operating condition. The concentration of a crude broth is beneficial in increasing the recovery yield of the desired protein. Using a constant amount of the affinity adsorbent, the recovery yield can be enhanced by decreasing the solution volume in the stirred tank due to the increase of the adsorbent weight fraction. It was found that the lumped kinetic model was simple and useful in analyzing the whole affinity filtration process.

Shenjincao (Palhinhaea cernua) injection for treatment of experimental silicosis of rats.

Shenjincao injection is a traditional Chinese medicine prepared from Palhinhaea cernua (L.) A. Franco et Vasc. by ultrafiltration. Its anti-silicosis action has been investigated both as a prophylactic and for treatment of the disease. Wistar rats were injected intra-tracheally with quartz dust and then divided randomly into groups-treatment and control prophylactic groups and treatment and control disease groups. After five days or eight weeks, respectively, the silica-exposed rats of the two treatment groups were injected intraperitoneally three times a week with shenjincao injection, dose 2.0 mL, for five weeks or 11 weeks, respectively. The rats were then dissected, and the ceruloplasmin content of the serum and the fresh weight, dry weight, collagen content and pathological grade of the lungs were measured. Compared with the corresponding exposed control groups for the same treatment periods the values of these parameters were reduced by 62.8% to 30.7% for rats in the prophylactic treatment group (P < 0.01 for all) and by 50.8% to 30.2% for the diseased group (P < 0.01 for all). The values for the disease-treatment group were also reduced by 37.9% to 25.9% compared with values for the exposed control group before treatment (P < 0.01 or P < 0.05). The effective coefficients for prophylactic treatment were 82.6% to 56.0%; for disease treatment they were 68.8% to 39.8%. These results show that shenjincao injection is efficacious against experimental silicosis not only when used prophylactically but also when used to treat the disease.

[Effects of sympathetic efferent in diabetic hyperalgesia in rat].

While sympathetic efferents and prostaglandins (PGs) play an important role in hyperalgesia of partial injury of peripheral nerves and inflammation, whether these are also involved in diabetic hyperalgesia is unknown. With intraperitoneal injection of 6-hydroxydopamine (6-OHDA) to eliminate effects of sympathetic postganglionic neurons (SPGNs) terminals, a model of diabetic rats with infused 6-OHDA could be set up by injection of streptozotocin (STZ). Nociceptive paw-withdrawal threshold (NPWT) and tail flick latency (TFL) in 6-OHDA/diabetic rats were not changed significantly in the following four weeks. However, in diabetic group rats, pain threshold was decreased significantly and accompanied by development of hyperalgesia. NPWT was significantly decreased with noradrenaline (NA) in diabetic hyperalgesic rats and increased with phentolamine or yohimbine, but not by prazocin. In the control rats, NPWT are not changed significantly by NA or phentolamine, and in 6-OHDA/diabetic rats, neither NA nor phentolamine also affected on NPWT significantly. However, NPWT may be significantly decreased by PGE1, PGE2 and PGD2 in both the control and the diabetic hyperalgesic rats. The above results suggested that SPGNs terminals are involved in hyperalgesia of diabetic rats, NA accelerated synthesis of PGs and released by way of presynaptic effect on alpha 2-adrenergic receptors at the SPGNs terminals, PGs, in turn, directly acted on the primary afferent nociceptors, producing hyperalgesia.

[Responsive properties of afferent units of the superficial branch of radial nerve in diabetic neuropathic patients].

By means of intraneural microrecording (INMR), changes of mechanical threshold and conduction velocity (CV) of A beta mechanical receptive units and A delta units of the superficial branch of the radial nerve and their response to sympathetic efferent were studied in 8 diabetic neuropathic patients. The results showed that the CV of these units was significantly decreased, and so was the mechanical threshold of A delta units. During the aftermath of excitation of the skin sympathetic nerve by mental arithmetics, 3/12 A delta units were turned to the active state with decreased mechanical threshold.

[Responsive property of afferent units in the caudal nerve of streptozotocin-diabetic rats].

In the present study, the responsive properties of afferent units in caudal nerve of streptozotocin (STZ) induced diabetic rats were investigated. It was found that some of the C and A delta units spontaneously discharged and their mechanical threshold was decreased significantly in diabetic rats. The response of the C units of diabetic and control rats to a 1 min sustained mechanical stimulus of threshold strength was similar, whereas, discharge frequencies during sustained suprathreshold mechanical stimulus were significantly greater in the C units of diabetic rats. After removal of threshold or suprathreshold mechanical stimulus, after discharges were also greater in C units of diabetic rats. Conduction velocity of C units, A delta units and A beta mechanical receptive units were also decreased in diabetic rats. However, there was no significant difference in the subunits of all kinds of receptive units between diabetic and control rats. The present data suggested that the C and A delta units with lowered mechanical threshold contribute to the decrease of the behavioral nociceptive threshold of diabetic rats and abnormal discharge of the C and A delta units may be a peripheral factor in hypersensitivity to painful stimuli and paresthesia of diabetic rats.

[Physiological properties of A delta and C fibres of superficial branch of radial nerve in human].

By means of intraneural micro recording (INMR) and intraneural microstimulation (INMS), properties of receptive units innervated by A delta and C fibers of the superficial branch of the radial nerve were measured on humans. Thresholds of 24 A delta units to mechanical stimulation varied significantly; their receptive fields ranged from a single point to 75.4 mm2, with one to four receptive sensitive zones. A delta units by INMS evoked tingling with projected fields varying between 1.2 and 97 mm2. Receptive fields and projected fields of these units overlapped each other and showed a proximodistal size gradient (P < 0.05). The results suggest that A delta units have accurate localizing capacity. Eleven nociceptive units had higher threshold to mechanical stimulation. INMS of C nociceptive units evoked burning pain. Both the receptive fields and projected fields of most of these units showed overlapping indicating that C nociceptive units, to some extent, can localize noxious stimulation.

[Clinical study on treatment of gastric ulcer with qingwei zhitong pill].

OBJECTIVE: To observe the therapeutical effect of Qingwei Zhitong pill (QWZTP) in treating gastric ulcer and clearance of the Helicobacter Pylori (HP). METHODS: Patients in the treated group (n = 60) and the control group (n = 60) were treated with QWZTP and Sifangwei tablet separately to observe the therapeutic effect of treatment on ulcer niche, TCM Syndromes and HP. RESULTS: The effective rate on ulcer niche evaluated by gastroscope was 86.67% in the treated group and 71.67% in the control group, the comparison between the two groups showed a significant difference, P < 0.05. The effective rate on improving TCM Syndrome in the two groups was 91.67% and 88.33% respectively, the difference between two groups was insignificant (P > 0.05). The HP clearance rate in the treated group was 47%. CONCLUSION: QWZTP has good effect in treating gastric ulcer and clearance of HP.

Purification of lysozyme by multistage affinity filtration.

A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified from a model system (a mixture of lysozyme and BSA) and a natural source (chicken egg white). From the chicken egg white, the three-stage affinity filtration increased the recovery yield of lysozyme from 61 to 96%, compared with the one-stage process. A mathematical model taking into account the film and interior diffusions of protein and eluant was developed for the modeling and analysis of the experimental data. Both the experimental and modeling results indicate that the multistage affinity filtration technique can be employed for the selective recovery of proteins.

Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML-RARalpha and PLZF-RARalpha oncoproteins.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations always involving the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes) in t(15;17) or t(11;17), respectively. APL in patients harboring t(15;17) responds well to retinoic acid (RA) treatment and chemotherapy, whereas t(11;17) APL responds poorly to both treatments, thus defining a distinct syndrome. Here, we show that RA, As(2)O(3), and RA + As(2)O(3) prolonged survival in either leukemic PML-RARalpha transgenic mice or nude mice transplanted with PML-RARalpha leukemic cells. RA + As(2)O(3) prolonged survival compared with treatment with either drug alone. In contrast, neither in PLZF-RARalpha transgenic mice nor in nude mice transplanted with PLZF-RARalpha cells did any of the three regimens induce complete disease remission. Unexpectedly, therapeutic doses of RA and RA + As(2)O(3) can induce, both in vivo and in vitro, the degradation of either PML-RARalpha or PLZF-RARalpha proteins, suggesting that the maintenance of the leukemic phenotype depends on the continuous presence of the former, but not the latter. Our findings lead to three major conclusions with relevant therapeutic implications: (i) the X-RARalpha oncoprotein directly determines response to treatment and plays a distinct role in the maintenance of the malignant phenotype; (ii) As(2)O(3) and/or As(2)O(3) + RA combination may be beneficial for the treatment of t(15;17) APL but not for t(11;17) APL; and (iii) therapeutic strategies aimed solely at degrading the X-RARalpha oncoprotein may not be effective in t(11;17) APL.

Two critical hits for promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.

Acute leukemia with promyelocytic features in PML/RARalpha transgenic mice.

Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations involving the retinoic acid receptor alpha (RARalpha) locus on chromosome 17. In the majority of cases, RARalpha translocates and fuses with the promyelocytic leukemia (PML) gene located on chromosome 15. The resulting fusion genes encode the two structurally unique PML/RARalpha and RARalpha/PML fusion proteins as well as aberrant PML gene products, the respective pathogenetic roles of which have not been elucidated. We have generated transgenic mice in which the PML/RARalpha fusion protein is specifically expressed in the myeloid-promyelocytic lineage. During their first year of life, all the PML/RARalpha transgenic mice have an abnormal hematopoiesis that can best be described as a myeloproliferative disorder. Between 12 and 14 months of age, 10% of them develop a form of acute leukemia with a differentiation block at the promyelocytic stage that closely mimics human APL even in its response to retinoic acid. Our results are conclusive in vivo evidence that PML/RARalpha plays a crucial role in the pathogenesis of APL.