Responses of hemopoietic precursors to 13-cis retinoic acid and 1,25 dihydroxyvitamin D3 in the myelodysplastic syndromes

To determine the effects of the "maturation-inducing" agents 13-cis retinoic acid and 1,25 dihydroxyvitamin D3 on marrow cells from normal individuals and patients with myelodysplastic syndromes (MDS), we assessed marrow hemopoietic clonogenicity and differentiation response patterns to these agents. These vitamins caused increased proliferation in vitro of normal clonogenic marrow myeloid precursor cells (CFU-GM), decreased erythroid precursors (BFU-E), and no change in multipotent stem cells (CFU-GEMM). Marrow hemopoietic colony-forming cell incidence was generally subnormal in the 22 MDS patients evaluated. In vitro exposure to both agents caused various patterns of alteration of MDS hemopoietic colony and cluster formation, with similar but more pronounced effects evoked by retinoic acid. In the vast majority of MDS patients, enhanced marrow clonal granulocyte-monocyte differentiation and decreased BFU-E growth were noted after in vitro exposure to these vitamins. Correlation of biological effects was demonstrated between in vivo changes of peripheral neutrophil counts and in vitro responses of myeloid precursors for ten MDS patients treated with an eight-week therapeutic course of retinoic acid. Cytogenetic analyses indicated persisting aneuploidy or coexisting normal and aneuploid karyotypes in the cultured MDS myeloid cells and (with one exception) in native marrow cells from the treated patients. The varying responses of the MDS cells may monitor differing proportions of normal versus leukemic marrow cells susceptible to proliferative and differentiative expression on exposure to these agents.     

Treatment of 34 patients with myelodysplastic syndromes with 13-CIS retinoic acid

Abstract: Thirty-four patients with myelodysplastic syndromes, 23 men and 11 women, aged between 47 and 80 years, with all types of myelodysplastic syndromes were treated with 13-cis-retinoic acid. The dose of retinoic acid ranged between 10 and 60 mg/m²/daily and was administered in combination with vitamin E to diminish side effects. The duration of treatment was 3 months to 5 years. Partial remission was achieved in 4 patients, 1 with RA type, 2 with RAEB and 1 with CMML. Survival ranged from 1 to 5 years. Patients who received retinoic acid developed mild side effects. In conclusion, the administration of 13-cis-retinoic acid improves the hematological picture in a small number of MDS patients (11.7%).     

Randomized study of 13-cis retinoic acid v placebo in the myelodysplastic disorders

A double-blind, placebo-controlled randomized trial of 13-cis retinoic acid was performed to determine if the drug has a therapeutic effect in patients with myelodysplastic syndromes (MDS). Sixty-eight evaluable patients with MDS were randomized to receive a single, daily oral dose of either 13-cis retinoic acid (13-CRA, 100 mg/m2) or matching placebo. Treatment was continued, when possible, for a period of 6 months. Determination of response to treatment was based on clinical course, repeat bone marrow biopsies, and aspirates and blood counts (CBC) with WBC differential, platelet, and reticulocyte numbers at specified intervals. No significant difference was noted between the two treatment groups in response to test drug (P = .66). One patient (3%) in the 13-CRA group and two patients (6%) in the placebo group had a minor response. Approximately 30% of patients in both groups had progression of their disease, and progression-free survival was nearly identical. Greater than 90% of the patients receiving 13-CRA developed mild or moderate skin toxicity that was reversible with decreasing or discontinuing the drug. Our study did not find that 13-CRA exerts a beneficial therapeutic effect in patients with MDS.     

A randomized trial of 13-cis retinoic acid with or without cytosine arabinoside in patients with the myelodysplastic syndrome

Ninety-eight consecutive patients with myelodysplastic syndrome were randomized to a treated or a control group, both receiving conventional supportive therapy. The treated group were given 13-cis-retinoic acid 20 mg/d if marrow blasts were less than or equal to 5% or cytosine arabinoside 10 mg/d subcutaneously on 6 d/week if marrow blasts were 6-30%, to which retinoic acid was added after 12 weeks. Serum levels of the drugs in the treated group were similar to those that would produce inhibition of CFU-GM growth in vitro. In patients in the low blast group receiving retinoic acid, myeloid surface antigens reverted from an abnormal to a normal pattern. Log rank analysis carried out after 25 months showed no significant difference in survival between the treated and control group, either in the total patient population or in the high and low blast groups considered separately. However, analysis of 39 non-sideroblastic patients with less than or equal to 5% blasts showed an increase in survival in the treated group.     

Dystrophy of the vulva locally treated with 13-cis retinoic acid.

Topical application of 13-cis retinoic acid resulted in complete disappearance of dystrophy of the vulva in 33 out of 53 patients, among them in 9 patients with recurrence after vulvectomy and in 14 patients with previous unsuccessful conservative treatment. Partial disappearance of dystrophy was seen in 15 patients, usually after 1 to 2 months of daily retinoid treatment. Side-effects of treatment (except for one) could be managed. Serum retinol level in patients was found to be lower as compared to that in healthy subjects. The results suggest that patients with chronic epithelial vulvar dystrophies could benefit from local retinol treatment, especially in cases with recurrence after vulvectomy and in cases with advanced dystrophies qualified until now for vulvectomy.     

All-trans retinoic acid in patients with myelodysplastic syndromes: results of a pilot study

Considering the beneficial effect of all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL), it has been speculated that ATRA might also be useful for treating other hematologic malignancies. To test this hypothesis, we performed a dose-escalating 3-month-trial of ATRA in 15 patients with primary or secondary myelodysplastic syndromes (MDS). Morphologic diagnoses were refractory anemia (RA) in 4, RA with ring sideroblasts (RARS) in 2, RA with excess blasts (RAEB) in 7, and RAEB in transformation (RAEB/T) in 2 cases. Patients included were required to have one or more of the following criteria: transfusion- dependent anemia, pronounced neutropenia (< or = 0.5 x 10(9)/L) or thrombocytopenia (< or = 20 x 10(9)/L), or increasing blast cells in the peripheral blood or bone marrow. Therapy was started at an ATRA dose of 30 mg/m2/d, administered orally as two doses of 15 mg/m2 every 12 hours. The retinoid dose was increased to 60 mg/m2/d after 4 weeks and to 90 mg/m2/d after 8 weeks. Among 14 patients assessable for response, none obtained a complete or partial remission. Three patients had a minor response, manifested by either reduction in transfusion requirements (2 patients) or increase in neutrophil and platelet counts (1 patient). During the study period, 5 patients progressed to more advanced stages of MDS or overt leukemia. Three patients with chromosomal abnormalities receiving ATRA for a period of 10 to 12 weeks retained their cytogenetic marker after completion of treatment. Side effects of ATRA primarily affected the skin and mucous membranes, with 13 of 15 patients having at least low-grade dermatologic toxicity. In 2 cases, treatment had to be prematurely stopped because of intolerable conjunctivitis or progressive neurologic symptoms. These data suggest that ATRA has little effect on MDS. The lack of response of MDS patients, as compared with those with APL, may be attributed to the absence of the t(15;17) translocation that seems to be a prerequisite for clinical efficacy of ATRA.     

1,25-Dihydroxyvitamin D3 up-regulates the 1,25-dihydroxyvitamin D3 receptor in vivo.

The level of mRNA encoding the 1,25-dihydroxyvitamin D3 receptor in the intestine of vitamin D-deficient rats given 1,25-dihydroxyvitamin D3 was determined by Northern blot analysis using a 32P-labeled cDNA probe to the 1,25-dihydroxyvitamin D3 receptor. mRNA levels increased 10-fold above deficiency levels at 6 and 12 hr after an intravenous dose of 1,25-dihydroxyvitamin D3, returning to predosing levels at 24 hr. Total receptor protein level determined by an immunoradiometric assay was increased 2-fold at 12 hr. No change in unoccupied receptor levels determined by ligand-binding assay was observed during this period. These results suggest that 1,25-dihydroxyvitamin D3 increases receptor mRNA and total receptor level to maintain constant levels of unoccupied receptor.     

1，25-二羟维生素D3治疗糖尿病肾病疗效观察

目的:探讨1,25-二羟维生素D_3治疗糖尿病肾病(DN)的疗效。方法:将60例DN患者随机分为试验组和对照组,每组30例。2组患者均接受常规降糖治疗,对照组患者口服厄贝沙坦150～300 mg/d;试验组患者口服厄贝沙坦150～300 mg/d,联合口服阿法骨化醇0.25μg,1次/d。记录并比较2组患者治疗前、后血糖化血红蛋白、肌酐等水平。结果:治疗3个月后试验组肌酐、尿N-乙酰-β-氨基葡萄糖苷酶、尿蛋白、血转化生长因子β_1均显著低于对照组(均P0.05)。结论:1,25-二羟维生素D_3对DN患者有肾保护作用。     

Differentiation of human myeloid leukemia cell line ML-1 induced by retinoic acid and 1,25-dihydroxyvitamin D3.

Human myeloblastic leukemia cell line ML-1 was induced to differentiate by 1 mumol/l all-trans-retinoic acid (RA) or by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). After 4-6 days of the induction several differentiation-associated characteristics were observed: (1) Ability to stimulate respiration burst in the ML-1 induced cells as detected by nitroblue tetrazolium (NBT) test or by chemiluminescence (CL). (The percentage of the NBT-positive cells was up to 99% in the RA-induced cells and up to 85% in the 1,25(OH)2D3-induced cells.) (2) Substantially higher phagocytosis of colloid iron, latex or Staphylococcus particles was found in the induced cells. (3) The 1,25(OH)2D3-induced ML-1 cells expressed the monocytic enzyme NaF-inhibitable alpha-naphthyl butyrate esterase and the surface monocytic antigen CD-14. (4) A majority of the induced cells lost the morphological features of blast cells; while the 1,25(OH)2D3-treated cells acquired certain features of monocyte-macrophage differentiation, the RA-treated cells displayed several granulocytic characteristics. (5) Cytofluorometric DNA assay after treatment of the cells with colcemid showed that the decline observed in the growth rate of the induced cells was connected with their arrest in G1/G0 phase of the cell cycle. The obtained results indicate granulocytic differentiation of the RA-induced ML-1 cells and monocyto/macrophage differentiation of the 1,25-(OH)2-D3 induced cells.     

13-cis-Retinoic acid treatment in patients with myelodysplastic syndrome

Twenty patients with primary myelodysplastic syndromes (16 refractory anemia without or with ringed sideroblasts, 2 refractory anemia with excess blasts, 2 refractory anemia with excess blasts in transformation) received 13-cis-retinoic acid at a dosage of 50-100 mg/m2/day for a minimum of 4 weeks. Twelve patients obtained an increase of hemoglobin levels greater than 1 g/dl and 7 showed an associated increase of granulocyte count greater than 50% of baseline values. No significant biochemical signs of dyslipidemia or liver damage were noted. A sustained response was noted only in refractory anemia without or with ringed sideroblasts and normal or hypercellular bone marrow. Five patients are still on therapy from 23 to 82 weeks without transfusion requirement and all have shown an improvement in performance status. We conclude that 13-retinoic acid may only be clinically useful in selected patients since in myelodysplastic syndromes with blast excess the drug does not seem to improve the course of the disease.     

Valproic acid and all-trans retinoic acid for the treatment of elderly patients with acute myeloid leukemia

Valproic acid (VPA) has been demonstrated to be able to inhibit histone deacetylase activity and to synergize with all-trans retinoic acid (ATRA) in inducing the differentiation of acute myeloid leukemia (AML) cells. A pilot study of the VPA/ATRA combination was performed in 11 elderly patients with de novo AML (median age, 82 years). Complete marrow response was observed in 3 patients, including 1 complete remission. Two additional patients had hematologic improvement.     

Induction of androgen receptor by 1alpha,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells

We have recently shown that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits proliferation of LNCaP cells, an androgen-responsive human prostate cancer cell line. Also, 1,25-(OH)2D3 increases androgen receptor (AR) abundance and enhances cellular responses to androgen in these cells. In the current study, we have investigated the mechanism by which 1,25-(OH)2D3 regulates AR gene expression and the involvement of AR in the 1,25-(OH)2D3- and 9-cis retinoic acid (RA)-mediated growth inhibition of LNCaP cells. Northern blot analyses demonstrated that the steady-state messenger RNA (mRNA) level of AR was significantly increased by 1,25-(OH)2D3 in a dose-dependent manner. Time-course experiments revealed that the increase of AR mRNA by 1,25-(OH)2D3 exhibited delayed kinetics. In response to 1,25-(OH)2D3, AR mRNA levels were first detected to rise at 8 h and reached a maximal induction of 10-fold over the untreated control at 48 h; the effect was sustained at 72 h. Furthermore, the induction of AR mRNA by 1,25-(OH)2D3 was completely abolished by incubation of cells with cycloheximide, a protein synthesis inhibitor. 1,25-(OH)2D3 was unable to induce expression of an AR promoter-luciferase reporter. Together, these findings indicate that the stimulatory effect of 1,25-(OH)2D3 on AR gene expression is indirect. Western blot analyses showed an increase of AR protein in 1,25-(OH)2D3-treated cells. This increased expression of AR was followed by 1,25-(OH)2D3-induced inhibition of growth in LNCaP cells. Similar to 1,25-(OH)2D3, 9-cis RA also induced AR mRNA expression, and the effect of both hormones was additive. Moreover, 1,25-(OH)2D3 and 9-cis RA acted synergistically to inhibit LNCaP cell growth. These antiproliferative effects of 1,25-(OH)2D3 and 9-cis RA, alone or in combination, were blocked by the pure AR antagonist, Casodex. In conclusion, our results demonstrate that growth inhibition of LNCaP cells by 1,25-(OH)2D3 and 9-cis RA is mediated by an AR-dependent mechanism and preceded by the induction of AR gene expression. This finding, that differentiating agents such as vitamin D and A derivatives are potent inducers of AR, may have clinical implications in the treatment of prostate cancer.     

Sustained response to recombinant human erythropoietin and intermittent all-trans retinoic acid in patients with myelodysplastic syndromes

In vitro studies suggest that all-trans retinoic acid (ATRA) synergizes with erythropoietin (EPO) for the stimulation of hematopoiesis in patients with myelodysplastic syndrome (MDS). A clinical trial was performed to evaluate whether a combination of these agents was effective in relieving the cytopenias associated with MDS. Twenty-seven patients with low- or intermediate-risk MDS were enrolled in a 12-week study. ATRA was administered orally at the dose of 80 mg/m(2) per day in 2 divided doses for 7 consecutive days every other week. Recombinant human EPO was given subcutaneously 3 times a week. The EPO dose was initiated at 150 U/kg and was increased to 300 U/kg if after 6 weeks there was no or there was suboptimal erythroid response. Patients who responded to therapy were continued on ATRA and EPO at the same doses for 6 additional months (extension phase). Further treatment was given to patients with a continued response. Clinically significant erythroid responses with increases of hemoglobin levels of at least 1 g/dL or reduction of transfusion needs were seen in 13 (48%) patients, with 4 showing improved responses after dose escalation of EPO. Ten (37%) patients displayed continued responses during 6 months of extended treatment, and 7 (26%) are still responsive after a follow-up period of 13 months. Neutrophil responses were observed in 5 of 12 patients with neutropenia, and platelet responses were observed in 6 of 9 patients with thrombocytopenia. Three patients displayed trilineage responses that were sustained during continuation therapy. Side effects were observed in all patients but were of mild entity and did not require discontinuation of therapy. It is concluded that the combination ATRA + EPO is an effective and well-tolerated treatment for patients with low- and intermediate-risk MDS. The optimal ATRA and EPO schedule and the role of maintenance treatment remain to be determined and warrant further investigation.     

13-cis retinoic acid augments the production of macrophages in mouse bone marrow cultures stimulated with interleukin 3

Mouse bone marrow cells in liquid culture with interleukin 3 generate nonadherent granulocytes, mast cells, and macrophages. The addition of 13-cis retinoic acid (13cRA) (10(-8)-10(-6) M) enhanced proliferation of the nonadherent cells, and concentrations greater than 5 x 10(-7) M stimulated a sixfold increase in adherent macrophages. Four-color flow cytometry was used to identify the lineages present using the following antibodies: MAC1 (granulocytes and macrophages), F4/80 (macrophages), B54.2 (mast cells), and H12 (anti-Thy1.2 to identify myeloid precursors). This analysis demonstrated a twofold increase in MAC1+ F4/80+ cells, which were sorted and identified morphologically as macrophages. 13cRA also increased by 60%-95% the numbers of colony-forming cells responsive to interleukin 3 (IL-3) and macrophage colony-stimulating factor (M-CSF) but did not significantly change the colony-forming cells responsive to granulocyte-macrophage colony-stimulating factor (GM-CSF). These data suggest that 13cRA increases the production of macrophages by modulating the commitment of IL-3-expanded progenitor cells to the macrophage lineage.