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Biomedical subjects

R C Ribeiro

Publications and source records attributed to R C Ribeiro.

At least 19 recordsLinked to original sources
PubMed2000

Traumatic lumbar puncture at diagnosis adversely affects outcome in childhood acute lymphoblastic leukemia.

The effect of traumatic lumbar puncture at the time of initial diagnostic workup on treatment outcome in children with newly diagnosed acute lymphoblastic leukemia (ALL) was investigated. The findings of the first 2 lumbar punctures performed on 546 patients with newly diagnosed ALL treated on 2 consecutive front-line studies (1984-1991) at St Jude Children's Research Hospital were retrospectively reviewed. Lumbar punctures were performed at the time of diagnosis and again for the instillation of first intrathecal chemotherapy. The event-free survival (EFS) experience for patients with 1 cerebrospinal fluid (CSF) sample contaminated with blast cells was worse than that for patients with no contaminated CSF samples (P =.026); that of patients with 2 consecutive contaminated CSF samples was particularly poor (5-year EFS = 46 +/- 9%). In a Cox multiple regression analysis, the strongest prognostic indicator was 2 consecutive contaminated CSF samples, with a hazard ratio of 2.39 (95% confidence interval, 1. 36-4.20). These data indicate that contamination of CSF with circulating leukemic blast cells during diagnostic lumbar puncture can adversely affect the treatment outcome of children with ALL and is an indication to intensify intrathecal therapy.

Adolescent↗
PubMed2000

Safety of lumbar puncture for children with acute lymphoblastic leukemia and thrombocytopenia.

CONTEXT: Patients with thrombocytopenia are at risk for spontaneous or procedure-related hemorrhage. Whether such patients can safely undergo lumbar puncture (LP) without prophylactic platelet transfusion is unknown. OBJECTIVE: To determine whether an association exists between thrombocytopenia and LP complications among children with acute lymphoblastic leukemia. DESIGN, SETTING, AND PATIENTS: Retrospective review of the records of 958 consecutive children (median age, 5.5 years) with newly diagnosed acute lymphoblastic leukemia who were treated at a pediatric cancer center between February 1984 and July 1998. INTERVENTIONS: All patients underwent a diagnostic LP followed by a median of 4 LPs to instill intrathecal chemotherapy. MAIN OUTCOME MEASURE: Serious complications of LP occurring during the remission induction and consolidation treatment periods (when thrombocytopenia is likely to occur), defined as any neurologic, infectious, or hemorrhagic problems related to the procedure, reported by platelet count at the time of the procedure. RESULTS: Of the 5223 LPs evaluated, 29 were performed at platelet counts of 10 x 10(9)/L or less, 170 at platelet counts of 11 to 20 x 10(9)/L, and 742 at platelet counts of 21 to 50 x 10(9)/L. No serious complications were encountered, regardless of the platelet count. The 95% confidence interval for the proportion of serious complications in the 199 patients with platelet counts of 20 x 10(9)/L or less was 0% to 1.75% and that for the 941 patients with platelet counts of 50 x 10(9)/L or less was 0% to 0.37%. CONCLUSIONS: In our study of children undergoing remission induction or consolidation therapy for acute lymphoblastic leukemia, serious complications of LP were not observed, regardless of platelet count. Prophylactic platelet transfusion is not necessary in children with platelet counts higher than 10 x 10(9)/L. Due to the small number of patients in our study with platelet counts of 10 x 10(9)/L or less, conclusions cannot yet be drawn for such patients. JAMA. 2000;284:2222-2224.

Adolescent↗
PubMed2000

Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia.

By using rapid flow cytometric techniques capable of detecting one leukemic cell in 10(4) normal cells, we prospectively studied minimal residual disease (MRD) in 195 children with newly diagnosed acute lymphoblastic leukemia (ALL) in clinical remission. Bone marrow aspirates (n = 629) were collected at the end of remission induction therapy and at 3 intervals thereafter. Detectable MRD (ie, > or = 0.01% leukemic mononuclear cells) at each time point was associated with a higher relapse rate (P < .001); patients with high levels of MRD at the end of the induction phase (> or = 1%) or at week 14 of continuation therapy (> or = 0.1%) had a particularly poor outcome. The predictive strength of MRD remained significant even after adjusting for adverse presenting features, excluding patients at very high or very low risk of relapse from the analysis, and considering levels of peripheral blood lymphoblasts at day 7 and day 10 of induction therapy. The incidence of relapse among patients with MRD at the end of the induction phase was 68% +/- 16% (SE) if they remained with MRD through week 14 of continuation therapy, compared with 7% +/- 7% if MRD became undetectable (P = .035). The persistence of MRD until week 32 was highly predictive of relapse (all 4 MRD(+) patients relapsed vs 2 of the 8 who converted to undetectable MRD status; P = .021). Sequential monitoring of MRD by the method described here provides highly significant, independent prognostic information in children with ALL. Recent improvements in this flow cytometric assay have made it applicable to more than 90% of all new patients. (Blood. 2000;96:2691-2696)

Adolescent↗
PubMed2000

Late effects of treatment in survivors of childhood acute myeloid leukemia.

PURPOSE: To investigate the incidence of and risk factors for late sequelae of treatment in patients who survived for more than 10 years after the diagnosis of childhood acute myeloid leukemia (AML). PATIENTS AND METHODS: Of 77 survivors (median follow-up duration, 16. 7 years), 44 (group A) had received chemotherapy, 18 (group B) had received chemotherapy and cranial irradiation, and 15 (group C) had received chemotherapy, total-body irradiation, and allogeneic bone marrow transplantation. Late complications, tobacco use, and health insurance status were assessed. RESULTS: Growth abnormalities were found in 51% of survivors, neurocognitive abnormalities in 30%, transfusion-acquired hepatitis in 28%, endocrine abnormalities in 16%, cataracts in 12%, and cardiac abnormalities in 8%. Younger age at the time of diagnosis or initiation of radiation therapy, higher dose of radiation, and treatment in groups B and C were risk factors for the development of academic difficulties and greater decrease in height Z: score. In addition, treatment in group C was a risk factor for a greater decrease in weight Z: score and the development of growth-hormone deficiency, hypothyroidism, hypogonadism, infertility, and cataracts. The estimated cumulative risk of a second malignancy at 20 years after diagnosis was 1.8% (95% confidence interval, 0.3% to 11.8%). Twenty-two patients (29%) were smokers, and 11 (14%) had no medical insurance at the time of last follow-up. CONCLUSION: Late sequelae are common in long-term survivors of childhood AML. Our findings should be useful in defining areas for surveillance of and intervention for late sequelae and in assessing the risk of individual late effects on the basis of age and history of treatment.

Acute Disease↗
PubMed2000

Coactivator-vitamin D receptor interactions mediate inhibition of the atrial natriuretic peptide promoter.

We have discovered a role for coactivators binding to the AF-2 surface of the vitamin D receptor (VDR) in its negative effects on gene transcription. We tested nine amino acid residues (Ser(235), Ile(242), Lys(246), Asp(253), Ile(260), Leu(263), Leu(417), Leu(419), and Glu(420)) in human VDR which, based on homology to the human thyroid hormone receptor, would be predicted to lie in or near the coactivator-binding site. Mutation of six of these residues in VDR resulted in loss of both the activation (assessed with a transfected DR3 TK luciferase reporter) and inhibition (assessed with an hANPCAT reporter) functions of the receptor when tested in cultured neonatal rat atrial myocytes and HeLa cells. Collectively, these mutations also suppressed association of VDR with the coactivators GRIP1 and steroid receptor coactivator 1 in vitro but had little or no effect on ligand binding, heterodimerization with the retinoid X receptor, or association with a VDR-specific DNA recognition element. Co-transfection with GRIP1 or steroid receptor coactivator 1 amplified both the positive and negative responses to wild type VDR but had little or no effect on the functionally impaired mutants described above. The interaction between VDR and GRIP1 proved to be heavily dependent upon the integrity of nuclear box III in the latter protein. Mutations in this region of GRIP1 impaired its ability to associate with VDR in vitro and to amplify VDR activity in intact cells. These studies establish a role for coactivators recruited to the same receptor surface in both the activating and inhibitory activity of the liganded receptor.

Amino Acid Sequence↗
PubMed2000

Splenic rupture in children with hematologic malignancies.

BACKGROUND: Splenic rupture is an uncommon but life-threatening complication of leukemias and lymphomas, and is reported mostly in adults. The authors investigated the frequency with which splenic rupture is diagnosed in pediatric patients with hematologic malignancies and reviewed its clinical profile and outcome. METHODS: The data base of St. Jude Children's Research Hospital was searched for cases coded as splenic laceration or rupture, splenic infarction, or splenectomy in patients diagnosed with lymphoma or leukemia between January 1962 and December 1997. The medical records of patients with histopathologic or radiologic evidence of splenic rupture were reviewed. The time spanned by the study was divided into early (1962-1990) and recent (1991-1997) eras to reflect the availability of modern diagnostic imaging techniques. RESULTS: Seven children experienced splenic rupture. They were between ages 5-17 years. There were four males and three females. Primary diagnoses included acute myeloid leukemia (four patients), acute lymphoblastic leukemia (two patients), and Hodgkin lymphoma (one patient). Five patients were diagnosed in the recent era and two in the early era. Four patients had radiologic or bacteriologic evidence of fungal infection concomitant with the splenic event. Of five deaths, only two were related causally to splenic rupture; these occurred in the early era. All seven acute episodes of splenic rupture were managed conservatively without surgery. CONCLUSIONS: The overall frequency with which splenic rupture was detected in children with hematologic malignancy at the study institution was 0.18%. In the recent era, the frequency of detection was 9-fold higher (0.55%) than that of the early era (0.06%). Improved imaging techniques and increased utilization of imaging studies may account for the increased incidental detection of "preclinical" splenic rupture. Adolescent age group, acute myeloid leukemia (especially acute promyelocytic leukemia), a high leukocyte count, thrombocytopenia, and coagulopathy may predispose children with leukemia to pathologic splenic rupture. Fungal infection frequently was associated with splenic rupture and may play a role in its pathogenesis.

Adolescent↗
PubMed2000

Imaging findings in pediatric adrenocortical carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC), a tumor that is rare among children, causes clinically evident hormonal disturbances. Imaging methods are used to stage disease and to plan surgical resection. OBJECTIVE: To describe the findings of the various imaging methods used to evaluate ACC. MATERIALS AND METHODS: We reviewed the records of ten consecutive patients (mean age, 8.1 years) who presented from 1987 to 1998 with ACC. All patients underwent computed tomography (CT) scanning; five underwent magnetic resonance (MR) imaging; four underwent ultrasonography (US); and eight underwent radionuclide bone scans. RESULTS: Seven patients presented with signs of hormonally functional tumors. Typical imaging findings consisted of a large, well-defined suprarenal tumor, containing calcifications (seven patients) with a thin capsule and central necrosis or hemorrhage (six patients). The inferior vena cava (IVC) was compressed by tumor in three patients, and ultrasonography demonstrated invasion of the IVC wall in one of these. Three patients' bone scans showed that the primary tumor took up radioactive tracer. Spread to lungs or liver or both was demonstrated in six patients. CONCLUSIONS: CT, US and MR imaging are effective methods of imaging the primary tumor. Chest CT and bone scintigraphy should be performed to detect metastases. The presence of a thin tumor capsule, a stellate central zone of necrosis, and evidence of hormonal function help distinguish ACC from neuroblastoma.

Adolescent↗
PubMed2000

Late effects in survivors of infant leukemia.

Little is known about the incidence of and risk factor for late effects of infant leukemia. We evaluated 19 children with acute lymphoblastic leukemia and 15 with acute myeloid leukemia who were diagnosed at age 12 months or younger and have survived for more than 5 years after the diagnosis (median length of follow-up, 13 years; range, 5.7-29 years). Ten patients received chemotherapy alone (group A), 17 received chemotherapy and CNS-directed radiation therapy (CRT) (group B), and seven received chemotherapy, CRT and bone marrow transplantation (group C). The most frequently observed late sequelae included problems in growth (66% of survivors), learning (50%), hypothyroidism (15%), and pubertal development (12%). Cataract, cardiac and hearing abnormalities occurred in 6% of patients. Only eight patients (24%) survive without late effects. In comparison to patients in group A, patients in groups B and C had a higher incidence of having at least one late complication (P = 0.009), a greater decrease in height Z score at 5 years after diagnosis (P = 0.023), and a higher incidence of academic difficulties (P = 0.004). The estimated odds of academic difficulties increased by 18% (P = 0.032) for each month younger in age at the time of CRT. These results indicate that late sequelae are common in longterm survivors of infant leukemia and are often related to CRT and the patient's age at the time of CRT.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Treatment of childhood acute myelogenous leukemia with an intensive regimen (AML-87) that individualizes etoposide and cytarabine dosages: short- and long-term effects.

The purpose of this study was to assess the feasibility and efficacy of a treatment regimen for pediatric acute myelogenous leukemia (AML) that uses four rotating drug pairs and adjusts dosages of etoposide and cytarabine to target specific plasma concentrations. Thirty-one girls and 27 boys (median age, 9.7 years) with de novo AML were treated on the protocol. Six cycles of chemotherapy were planned. Cycles 1 to 4 comprised the drug combinations cytarabine plus etoposide, cytarabine plus daunomycin, etoposide plus amsacrine, and etoposide plus azacitidine, respectively. For cycles 5 and 6, the first two combinations were repeated. Dosages were adjusted to achieve plasma concentrations of 1.0 microM +/- 0.1 microM cytarabine and 30 microM +/- 0.3 microM etoposide. Forty-four patients (76%) entered complete remission. Of those, 24 have had relapses; 23 remain alive in first or subsequent remission. The 5-year event-free survival (EFS) estimate was 31.0% +/- 5.9%; the 5-year survival estimate was 41.4% +/- 6.3%. Six patients (10%) died of the toxic effects of therapy. Severe neutropenia occurred in all cycles. Long-term complications of therapy included hepatitis C, cardiac insufficiency, and hearing loss. Adjustment of cytarabine and etoposide dosage was feasible for achieving targeted plasma drug concentrations; however, the potential clinical efficacy of this approach was offset by substantial acute and long-term toxicity.

Adolescent↗
PubMed2000

Hypersensitivity or development of antibodies to asparaginase does not impact treatment outcome of childhood acute lymphoblastic leukemia.

PURPOSE: Development of antibodies and hypersensitivity to asparaginase are common and may attenuate asparaginase effect. Our aim was to determine the relationship between antiasparaginase antibodies or hypersensitivity reactions and event-free survival (EFS). PATIENTS AND METHODS: One hundred fifty-four children with acute lymphoblastic leukemia received Escherichia coli asparaginase 10,000 IU/m(2) intramuscularly three times weekly for nine doses during multiagent induction and reinduction phases and for seven monthly doses during continuation treatment. Erwinia asparaginase was used in case of clinical hypersensitivity to E coli but not for subclinical development of antibodies. Plasma antiasparaginase antibody concentrations were measured on day 29 of induction in 152 patients. RESULTS: Antibodies were detectable in 54 patients (35.5%), of whom 30 (55.6%) exhibited hypersensitivity to asparaginase. Of the 98 patients who had no detectable antibodies, 18 (18.4%) had allergic reactions. Patients with antibodies were more likely to have a reaction than those without antibodies (P <.001). Among the 50 patients who experienced allergic reactions (including two for whom antibodies were not measured), 36 (72.0%) were subsequently given Erwinia asparaginase; seven (19.4%) reacted to this preparation. EFS did not differ among patients who did and did not have antibodies (P =.54), with 4-year EFS (+/- 1 SE) of 83% +/- 6% and 76% +/- 5%, respectively. Similarly, EFS did not differ among patients who did and did not develop allergic reactions (P =.68), with 4-year estimates of 82% +/- 6% and 78% +/- 5%, respectively. CONCLUSION: In this setting, in which most patients with allergy were switched to another preparation, there was no adverse prognostic impact of clinical or subclinical allergy to asparaginase.

Adolescent↗
PubMed2000

Long-term outcome of 25 children and adolescents with severe aplastic anemia treated with antithymocyte globulin.

Severe aplastic anemia (SAA) is probably an immune-mediated disorder, and immunosuppressive therapy is recommended for patients with no available donor for bone marrow transplant. Between October 1984 and November 1987, 25 consecutive children and adolescents with SAA with no HLA-compatible marrow donor received equine antithymocyte globulin (ATG) (15 mg kg-1 day-1) for 10 days. The patients were evaluated 6 weeks, 6 months, and 12 months after starting ATG treatment. Thereafter, patients were evaluated yearly until July 1998. Median age was 10 years (range, 1.5-20 years), granulocyte counts on referral ranged from 0.032 to 1.4 x 10(9)/l (median 0.256 x 10(9)/l), and 12 patients had granulocyte counts <0.2 x 10(9)/l. At a median follow-up of 9.6 years (range, 8.6-11.8 years), 10 patients (40%) remained alive with good marrow function. No morphologic evidence of hematological clonal disorders has been observed, although two patients probably have acquired clonal chromosomal abnormalities (trisomy 8 and del(6)q21, respectively). Responses to ATG were observed between 6 weeks and 6 months from the start of treatment in 60% of evaluable patients. The response rate was not different in patients whose granulocyte count at diagnosis was <0.2 x 10(9)/l, or in those who were <10 years of age. This study supports the view that, when compared with supportive measures, ATG is an effective treatment for children or adolescents with SAA. Although these results are inferior to those reported for marrow transplantation or more intensive immunosuppressive regimens, these patients who responded to ATG are long-term survivors with stable peripheral blood counts and a low rate of relapse.

Adolescent↗
PubMed2000

Amplification of 9q34 in childhood adrenocortical tumors: a specific feature unrelated to ethnic origin or living conditions.

Adrenocortical tumors (ACT) in children under 15 years of age exhibit some clinical and biological features distinct from ACT in adults. Cell proliferation, hypertrophy and cell death in adrenal cortex during the last months of gestation and the immediate postnatal period seem to be critical for the origin of ACT in children. Studies with large numbers of patients with childhood ACT have indicated a median age at diagnosis of about 4 years. In our institution, the median age was 3 years and 5 months, while the median age for first signs and symptoms was 2 years and 5 months (N = 72). Using the comparative genomic hybridization technique, we have reported a high frequency of 9q34 amplification in adenomas and carcinomas. This finding has been confirmed more recently by investigators in England. The lower socioeconomic status, the distinctive ethnic groups and all the regional differences in Southern Brazil in relation to patients in England indicate that these differences are not important to determine 9q34 amplification. Candidate amplified genes mapped to this locus are currently being investigated and Southern blot results obtained so far have discarded amplification of the abl oncogene. Amplification of 9q34 has not been found to be related to tumor size, staging, or malignant histopathological features, nor does it seem to be responsible for the higher incidence of ACT observed in Southern Brazil, but could be related to an ACT from embryonic origin.

Adenoma↗
PubMed2000

Adrenocortical tumors in children.

Childhood adrenocortical tumors (ACT) are rare. In the USA, only about 25 new cases occur each year. In Southern Brazil, however, approximately 10 times that many cases are diagnosed each year. Most cases occur in the contiguous states of São Paulo and Paraná. The cause of this higher rate has not been identified. Familial genetic predisposition to cancer (p53 mutations) and selected genetic syndromes (Beckwith-Wiedemann syndrome) have been associated with childhood ACT in general but not with the Brazilian counterpart. Most of the affected children are young girls with classic endocrine syndromes (virilizing and/or Cushing). Levels of urinary 17-ketosteroids and plasma dehydroepiandrosterone sulfate (DHEA-S), which are abnormal in approximately 90% of the cases, provide the pivotal clue to a diagnosis of ACT. Typical imaging findings of pediatric ACT consist of a large, well-defined suprarenal tumor containing calcifications with a thin capsule and central necrosis or hemorrhage. The pathologic classification of pediatric ACT is troublesome. Even an experienced pathologist can find it difficult to differentiate carcinoma from adenoma. Surgery is the single most important procedure in the successful treatment of ACT. The role of chemotherapy in the management of childhood ACT has not been established although occasional tumors are responsive to mitotane or cisplatin-containing regimens. Because of the heterogeneity and rarity of the disease, prognostic factors have been difficult to establish in pediatric ACT. Patients with incomplete tumor resection or with metastatic disease at diagnosis have a dismal prognosis. In patients with localized and completely resected tumors, the size of the tumor has predictive value. Patients with large tumors have a much higher relapse rate than those with small tumors.

Adenoma↗
PubMed1999

Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators.

The tumor necrosis factor-alpha (TNF-alpha) promoter was used to explore the molecular mechanisms of estradiol (E(2))-dependent repression of gene transcription. E(2) inhibited basal activity and abolished TNF-alpha activation of the TNF-alpha promoter. The E(2)-inhibitory element was mapped to the -125 to -82 region of the TNF-alpha promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) beta is more potent than ERalpha at repressing the -1044 TNF-alpha promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERbeta, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E(2)-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltransferase gene locus.

BACKGROUND: Patients with acute lymphoblastic leukemia are often treated with 6-mercaptopurine, and those with homozygous deficiency in thiopurine S-methyltransferase (TPMT) enzyme activity have an extreme sensitivity to this drug as a result of the accumulation of higher cellular concentrations of thioguanine nucleotides. We studied the metabolism, dose requirements, and tolerance of 6-mercaptopurine among patients with different TPMT phenotypes. METHODS: We compared, by use of statistical modeling, 6-mercaptopurine pharmacology and tolerance in 180 patients who achieved remission on St. Jude Children's Research Hospital Protocol Total XII composed of weekly methotrexate (40 mg/m(2)) and daily oral 6-mercaptopurine (75 mg/m(2)) given for 2.5 years, interrupted every 6 weeks during the first year for treatment with either high-dose methotrexate or teniposide plus cytarabine. Statistical tests were two-sided. RESULTS: Erythrocyte concentrations of thioguanine nucleotides (pmol/8 x 10(8) erythrocytes) were inversely related to TPMT enzyme activity (P<.01), with averages (+/- standard deviations) of 417 (+/-179), 963 (+/-752), and 3565 (+/-1282) in TPMT homozygous wild-type (n = 161), heterozygous (n = 17), and homozygous-deficient (n = 2) patients, respectively. There was complete concordance between TPMT genotype and phenotype in a subset of 28 patients for whom TPMT genotype was determined. There were no sex differences in thioguanine nucleotide concentrations (P =.24), TPMT enzyme activity (P =.22), or average weekly prescribed dose of 6-mercaptopurine (P=.49). The cumulative incidence of 6-mercaptopurine dose reductions due to toxicity was highest among patients homozygous for mutant TPMT (100%), intermediate among heterozygous patients (35%), and lowest among wild-type patients (7%) (P<.001), with average (+/- standard deviation) final weekly 6-mercaptopurine doses of 72 (+/-60), 449 (+/-160), and 528 (+/-90) mg/m(2), respectively. Lowering doses of 6-mercaptopurine in TPMT heterozygotes and in deficient patients allowed administration of full protocol doses of other chemotherapy while maintaining high thioguanine nucleotide concentrations. CONCLUSION: We conclude that genetic polymorphism in TPMT is an important determinant of mercaptopurine toxicity, even among patients who are heterozygous for this trait.

Antimetabolites, Antineoplastic↗
PubMed1999

Vitamin D-dependent suppression of human atrial natriuretic peptide gene promoter activity requires heterodimer assembly.

Crystallographic structures of the ligand-binding domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues that participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor (hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced binding to glutathione S-transferase-RXRalpha in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXRalpha on DR-3 (a direct repeat of AGGTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR effected a 3-fold induction of DR-3-dependent thymidine kinase-luciferase activity in cultured neonatal rat atrial myocytes, an effect that was increased to 8-9-fold by cotransfected hRXRalpha. Induction by K386A, in the presence or absence of RXRalpha, was only slightly lower than that seen with wild type VDR. On the other hand, I384R/Q385R alone displayed no stimulatory activity and less than 2-fold induction in the presence of hRXRalpha. Qualitatively similar findings were observed with the negative regulation of the human atrial natriuretic peptide gene promoter by these mutants. Collectively, these studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.

Amino Acid Sequence↗