Dental abnormalities in Schimke immuno-osseous dysplasia.

Described for the first time in 1971, Schimke immuno-osseous dysplasia (SIOD) is an autosomal-recessive multisystem disorder that is caused by bi-allelic mutations of SMARCAL1, which encodes a DNA annealing helicase. To define better the dental anomalies of SIOD, we reviewed the records from SIOD patients with identified bi-allelic SMARCAL1 mutations, and we found that 66.0% had microdontia, hypodontia, or malformed deciduous and permanent molars. Immunohistochemical analyses showed expression of SMARCAL1 in all developing teeth, raising the possibility that the malformations are cell-autonomous consequences of SMARCAL1 deficiency. We also found that stimulation of cultured skin fibroblasts from SIOD patients with the tooth morphogens WNT3A, BMP4, and TGFß1 identified altered transcriptional responses, raising the hypothesis that the dental malformations arise in part from altered responses to developmental morphogens. To the best of our knowledge, this is the first systematic study of the dental anomalies associated with SIOD.

Glomerular involvement in myelodysplastic syndromes.

Several reports have documented various forms of glomerular diseases in adults with myelodysplastic syndromes (MDS), but similar reports in children are lacking. We describe two children with MDS-associated steroid-responsive nephrotic syndrome (NS). Patient 1, who had MDS with myelofibrosis, presented with hepatosplenomegaly, pancytopenia, chronic hepatitis, moderate proteinuria, hypocomplementemia and elevated ANA titer. During initial prednisone treatment proteinuria markedly diminished and partial but transient hematological improvement occurred. Relapse subsequently occurred that manifested by overt NS and pancytopenia. High doses of prednisolone led to remission of the renal disease, but hematological remission did not occur. Persisting pancytopenia and repeated infections terminated in sepsis, 2 years after the onset of the MDS. Patient 2, who had refractory anemia with clonal monosomy 19, presented with bowel disease, hepatosplenomegaly, anemia and non-organ-specific autoantibodies. Prednisone led to both clinical and hematological remission. The hematologic disease relapsed 12 months later, when nephrotic-range proteinuria, hematuria and mild azotemia were also found. Corticosteroid treatment led to long-lasting renal and hematologic remission, maintained by a small dosage of prednisone. In both patients, renal biopsy findings were consistent with those seen in idiopathic NS. A Medline search disclosed 16 cases of glomerulopathy in the course of MDS in adult patients. Clinical features included NS, usually accompanied by renal insufficiency with acute, chronic, or rapidly progressive glomerulonephritis. On biopsy, membranous nephropathy, crescentic or mesangial proliferative glomerulonephritis, and AL amyloidosis were found. We conclude: (1) that glomerular disease may be present and should be searched for in patients with MDS and (2) that MDS can be added to the list of rare conditions associated with corticosteroid-responsive NS in children.

[Acute tubulointerstitial nephritis in children]. / Akutni tubulointersticijumski nefritis kod dece.

UNLABELLED: Acute tubulointerstitial nephritis (ATIN) is a rare renal disorder in children. Patients usually present non-specific symptoms and signs so that the diagnosis of ATIN is often delayed. The disease may be infection- or drug-induced or it may occur without a known cause. Early recognition and appropriated therapy usually lead to an excellent prognosis. The aim of the study was to describe clinical and laboratory findings and the course of ATIN in 21 patients, that are typical enough to enable early recognition of the disease as it is potentially reversible. METHODS: Between 1986 and 1997 we observed 21 patients, aged 7-16 years (mean, 12.8), with acute tubulointerstitial nephritis, including eight with tubulointerstitial nephritis and uveitis (TINU syndrome). Laboratory studies included urinalysis, complete blood count, erytrocyte sedimentation rate (ESR), plasma creatinine, glomerular filtration rate (GFR), electrolytes, proteins, IgG, C3, C4 antinuclear-antibodies (ANA), antistreptolysin-O and antibodies to hantaviruses. Renal ultrasound was done in all patients. Renal biopsy was performed in 5 children. RESULTS: All children had previously been healthy. The symptoms of ATIN developed within a few days (Table 1). The most common initial symptoms were fatigue, fever, gastrointestinal disturbances, anorexia and weight loss. Less common complaints included headache, arthralgias and maculopapular rash. On addmition no patient had hypertension, oedema or evidence of acute infection. ESR, plasma urea and creatinine concentrations were increased in all, plasma proteins and IgG levels in the majority of patients. ANA were negative in 15 pts in whom this analysis was performed; C3 and C4 levels were normal. In two children recent strepococcal and in the other 6 hantavirus infection was serologicaly proved. All patients had non-oliguric acute renal failure (ARF): GFR was 21.7 +/- 8 9 in 14 pts and 67 +/- 9.7 in 7 pts. Low urine specific gravity (1003-1014), mild proteinuria (0.3-0.4 g/24 h), leukocyturia and/or haematuria were found in all patients; glycosuria, aminoaciduria and decreased tubular reaposrption of phosphate (TRP) were found in 12/21, 9/21 and 9/14 patients, respectively. Urine cultures were negative in all children. Renal US demonstrated enlarged hyperechoic kidneys in 11 pts, in remaining 10 pts no abnormalities were found. Renal biopsy, performed in 5 children, confirmed the diagnosis of ATIN. Eight patients subsequently developed anterior uveitis as part of TINU syndrome. Treatment included supportive therapy in all and six patients received prednisolone for 4-8 weeks (40-60 mg/m2/24 h for 10-14 days with subsequent reduction of dose over several weeks). Anterior uveitis was successfully treated with topical steroids. Renal function completely recovered in all patients: GFR (109 +/- 22.6 ml/min) within a mean interval of 47 +/- 33 days, concentration ability within 2-12 (mean 4.5) months. DISCUSSION: Common clinical features of ATIN are non-oliguric acute renal failure of various degrees, signs of tubular dysfunction, proteinuria, haematuria, leukocyturia and absence of hypertension. All our patients had normal blood pressure, non-oliguric renal failure, proteinuria, hypostenuria and abnormal urinary sediment; about half of them had glycosuria and/or other signs of proximal tubular dysfunction. The most important causes of ATIN in children reported in literature are systemic infections and drugs. However, the cause of ATIN in our patients was assessed as being related to infection only in 8 patients and to diclofenac in one. No infection, drug, toxin or other cause could be identified in 4, as well as in 8 pts with TINU syndrome. The prognosis of ATIN in children is considered to be favourable, but some patients may develop chronic renal failure. Renal function completely recovered in all our patients; that is consistent with outcome data from the most reports. CONCLUSION: Acute tubulointerstitial nephritis is an important cause of ARF in children, its aetiology may be different and it carries an excellent prognosis. ATIN should be suspected in a child who presents typical, although non-specific symptoms and signs, associated with lukocyturia and/or microhaematuria, signs of tubular dysfunction and unexplained renal failure. The diagnosis can be verified at renal biopsy. Early recognition of the disease is important to remove possible aetiologic agents and to treat them before chronic lesions are present to avoid long-term renal damage.

[Schimke immuno-osseous dysplasia]. / Simkeova imunsko-kostana displazija.

Schimke immuno-osseous dysplasia (OMIM *242900) is a rare autosomal recessive disorder that affects primarily the bone, the immune system, the kidneys, the skin and the vascular system. The patients have intrauterine growth retardation, short stature with short neck and trunk, peculiar clinical phenotype: triangular face, broad nasal bridge, bulbous nasal tip, small palpebral fissures, long upper lip and low hairline. The characteristic features include spondyloepiphyseal dysplasia, hyperpigmented maculae, proteinuria with progressive renal failure, lymphopenia with recurrent infections and cerebral ischaemia. We describe a girl, 5 years old, with short-trunk type of dwarfism (height 75 cm, below 3rd centile), short neck, accentuated lumbal lordosis and protruding abdomen. The patient had peculiar face with a broad, depressed nasal bridge, bulbous nasal tip, and slightly elongated upper lip. The hair was thin and sparse. Numerous pigmented spots resembling lentigines were visible on the trunk and abdomen. Radiographs showed spondyloepiphyseal dysplasia. At the age of 2 years laboratory analyses showed normal growth hormone secretion, normal thyroid function tests, normal female karyotype and no mucopolisachariduria. Since the age of 4 years, several episodes of transitory right-sided hemiparesis with spontaneous recovery, were observed. Seizures occurred at 5 years of age, when the MRI brain imaging showed multiple areas of ischaemia. She also experienced transient nephrotic syndrome, lymphopenia and low IgG accompanied by septicaemia.

Manifestations and treatment of Schimke immuno-osseous dysplasia: 14 new cases and a review of the literature.

UNLABELLED: Schimke immuno-osseous dysplasia (SIOD) is a rare autosomal recessive spondylo-epiphyseal dysplasia. The characteristic features of SIOD include 1) short stature with hyperpigmented macules and an unusual facies, 2) proteinuria with progressive renal failure, 3) lymphopenia with recurrent infections, and 4) cerebral ischaemia. Although 25 patients have been reported with this disorder, the clinical course and phenotype of SIOD are not well characterized. This report summarizes the clinical findings, course and treatment of reported patients and includes 14 additional patients with SIOD. We emphasize the high incidence of cerebral ischaemia and ocular abnormalities, define the high incidence of thyroid dysfunction and blood cytopenia, and confirm the absence of effective and durable medical therapies. CONCLUSION: Schimke immuno-osseous dysplasia is a multi-system autosomal recessive disorder with variable expression that affects the skeletal, renal, immune, vascular, and haematopoietic systems. Medical therapy is limited especially for more severely affected individuals.

[Familial membranoproliferative glomerulonephritis]. / Familijarni membranoproliferacioni glomerulonefritis.

INTRODUCTION: Idiopathic membranoproliferative (mesangiocapillary) glomerulonephritis (MPGN) is a chronic, often progressive renal disease with variable clinical expression divided into three distinct morphological formes, now designated types I, II, and III, on the basis of immunofluorescent (IF) and ultrastructural appearances and complement profiles [1-5]. Several lines of evidences suggest a genetic basis for at least some cases of MPGN I and III. The extended haplotypes HLA-B8, DR3, SCO1, GLO2 were found to constitute 13% of the disease-associated haplotypes and 1% of control hyplotypes [8]. Significantly high percentage of those with MPGN I and III have inherited defects of the complement system [9]. Additional evidence for genetic factors is the rarity of the disease in blacks [10] and examples of MPGN occurring in families. The disease has been reported in siblings as well as in families with affected members spanning more than one generation [11-16]. Here we describe clinical and morphological features in two siblings affected by MPGN and present complement and HLA typing studies done in patients and their parents. A review of familial MPGN I and III cases reported between 1981 and 1996 is made, and genetic susceptibility factors for MPGN are discussed. SUBJECTS AND METHODS: Between 1976 and 1996 diagnosis of idiopathic MPGN was made in 24 patients, aged 516.5 years. The diagnosis was established after excluding systemic, liver and infectious disorders and malignant neoplasms. The MPGN type was confirmed by light microscopy, IF and electron microscopy studies of the renal biopsy tissues processed by standard techniques. One family with two siblings having MPGN was identified in our series. This family was examined for the presence of renal disease and an inherited complement defect. Laboratory evaluations of the patients and parents included complete urinalysis, serum protein, albumin, urea, creatinine and cholesterol levels and glomerular filtration rate (GFR) estimation. ANA, rheumatoid factors, cryoglobulins, immune complexes, HBV antigens and antibodies and anti-HCV antibodies were also determined. Haemolytic tests for CHSO (classical and alternative pathways) were carried out using standard techniques. The measurement of the various complement factors was carried out using a radial immunodiffusion technique with monospecific antisera (CIq, C2, C4, C3, C5, B, H). HLA-A, B, DR and DQ haplotypes were determined by microcytotoxicity assay of peripheral blood lymphocytes. RESULTS: Patient 1 (SC, male). Renal disease presented at the age of his five years with nephrotic syndrome resistant to corticosteroid treatment. Morphological features and serum complement profile suggested type I MPGN. Treatment consisted of alternate-day prednisone, followed by cyclosporine and then by cyclophosphamide. At the end of the follow-up lasting 5.5 years he had only moderate proteinuria. Patient 2 (MC, female). Proteinuria was revealed at the age of 3 years becoming progressive and leading to the nephrotic syndrome resistant to corticosteroids at the age of 6 years. Electronmicroscopy features were consistent with type III MPGN, although serum C3 and C4 levels remained normal all the time. The same treatment as in her brother was given but she remained persistently nephrotic and anaemic; hypertension developed when she was 6 years old and her renal function became to declaine at the age of 7.5 years. Detailed family studies failed to reveal any evidence of complement deficiencies or secondary cause of MPGN. Siblings had in common HLA-A24, B27, Bw4, DRI1, DRS2, and DQ3 antigens. DISCUSSION: In our patients clinical and morphological features are very similar and are consistent with diagnosis of MPGN, i.e. probably type I in the boy and type III in the girl. Although some extended haplotypes HLA-B8, DR3, SCO1, GLO2, were significantly more frequent (13%) than in controls (1%), and the patients with MPGN having this extended haplotype had

[Acute tubulointerstitial nephritis with uveitis]. / Akutni tubulointersticijumski nefritis s uveitisom.

Clinical features and natural course of acute tubulointerstial nephritis and uveitis (TINU syndrome) in five adolescent patients (3 girls and 2 boys), are presented. Initial nonspecific symptoms preceding nephropathy were anorexia, weight loss, fever and malaise. Inflammatory syndrome consisted of increased ESR, high plasma proteins and gamma globulins. Analysis of urine showed proteinuria and sterile leukocyturia. Laboratory features of tubular dysfunction and decreased GFR were found in all patients. Renal biopsy, which was performed in 2 pts, revealed acute interstitial nephritis. Anterior uveitis which appeared later, was successfully treated with topical steroids. Renal function completely recovered within a few month in four pts and markedly improved in one. Despite the fact that renal biopsy was not performed in all children, the combination of an acute nonoliguric renal failure without hypertension and signs of tubular dysfunction together with particular benign course, suggested acute idiopathic TINU syndrome.

Enhanced cancer growth in mice administered daily human-equivalent doses of some H1-antihistamines: predictive in vitro correlates.

BACKGROUND: Present studies of drug-induced tumor growth promotion have evolved from earlier investigations into the mechanism of action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy[ethanamine.HCl, a tamoxifen derivative which potently inhibits lymphocyte mitogenesis in vitro and stimulates tumor growth in vivo. It is thought that potency to bind to intracellular histamine receptors (HIC), some of which are on cytochromes P450, may correlate with tumor growth-promoting activity. PURPOSE: We assessed the effectiveness of five in vitro assays in predicting in vivo tumor growth stimulation by the H1-antihistamines loratadine, astemizole, cetirizine, hydroxyzine, and doxylamine. METHODS: Potency of each agent was ranked 1-5 in each of the following in vitro assays: 1) inhibition of [3H]histamine binding to microsomal HIC, 2) inhibition of histamine binding to microsomal P450, 3) inhibition of the P450-catalyzed demethylation of aminopyrine, 4) inhibition of lymphocyte mitogenesis, and 5) stimulation of tumor colony formation. An overall rank score was assigned to each drug and correlated with tumor growth stimulation in vivo. Two laboratories conducted in vivo studies in a blinded fashion. Female C57BL and C3H mice were given a subcutaneous injection on day 1 of syngeneic B16F10 melanoma cells (5 x 10(5)) or C-3 fibrosarcoma cells (1 x 10(5)), respectively. Mice were randomly assigned to treatment groups, then received a single, daily intraperitoneal injection of an estimated human-equivalent dose (or range of doses) of antihistamine or vehicle control for 18-21 days before being killed. Tumors were surgically removed and wet weights compared statistically among groups. RESULTS: The cumulative potency of each drug in affecting tumor growth or growth mechanisms in the five in vitro assays ranked as follows: Loratidine and astemizole ranked highest and were equally potent, followed in decreasing order by hydroxyzine, doxylamine, and cetirizine. A significant correlation (r = .97; P < .02) was observed between the rank order of potency of the antihistamines in all five in vitro assays and the rank order to enhance tumor growth in vivo: Loratidine and astemizole significantly (P < .001) promoted the growth of both melanoma and fibrosarcoma, hydroxyzine significantly (P < .001) promoted the growth of melanoma, while doxylamine and cetirizine did not promote the growth of either tumor. CONCLUSION: Data demonstrate that the in vitro assays predicted the propensity of each H1-antihistamine to stimulate cancer growth in vivo. IMPLICATION: These in vitro tests may prove valuable to screen potential tumor growth promoters.

[Lupus nephritis in children and adolescents (Part II: Prognosis)]. / Lupusni nefritis kod dece i adolesenata (prognoza bolesti).

In 27 children and adolescents (24 females, 3 males suffering from lupus nephritis the important role of renal pathohistologic changes in the evolution and outcome of the disease was analysed, and a five-year survival rate was calculated. The mean age at the onset of the disease was 12.1 +/- 1.9 (range: 0.7-0) years, the mean interval from onset to renal biopsy was 2.1 +/- 1.9 (0.1 - 7.0) years, ant the mean follow-up was 4.1 +/- 2.1 (1-8) years. The pathohistologic changes were classified according to the World Health Organisation criteria. At the end of the follow-up patients classified in Ib and II classes were without urinary abnormalities, i.e. without preoteinuria and/or haematuria. However, patients in class IV manifests nephrotic syndrome (2 pts), renal failure (3 pts) or proteinuria and heamaturia (6 pts), and two patients died. More severe clinical features and course of lupus nephiritis associated with infavourable outcome significantly correlated with WHO classes III and IV. A five-year survival rate (life-table method) of 88.5% is among the best results reported to date in children and adolescents with lupus nephritis.

Focal glomerular sclerosis and nephrotic syndrome in spondyloepiphyseal dysplasia.

The association of a spondyloepiphyseal dysplasia and disproportionate short stature with focal glomerular sclerosis is reported in two girls. Renal disease manifested by proteinuria at the age of 2.5 and 11 years, leading to treatment-resistant nephrotic syndrome over 15 and 45 months, respectively. One patient went into end-stage renal failure shortly after nephrotic syndrome developed, the other died from sepsis. The association of spondyloepiphyseal dysplasia and focal glomerular sclerosis with nephrotic syndrome may represent a distinct disease entity.

[Lupus nephritis in children and adolescents (clinical-morphologic correlation)]. / Lupusni nefritis kod dece i adolescenata (klinicko-morfoloska korelacija).

Clinical and laboratory features in 27 children and adolescents (24 females, 3 males) with lupus nephritis were analysed and correlated with pathohistologic findings obtained by renal biopsy. The mean age at the onset of the disease was 12.1 +/- 1.9 (range 0.1-7.0) years. In the 30% of patients nephritis was one of the presenting features of the disease. The most frequent signs of the lupus nephritis at the time of biopsy were proteinuria (9 pts), proteinuria and haematuria (9 pts), and nephrotic syndrome (8 pts), while 7 patients showed decreased glomerular filtration rate (GFR), and hypertension was present in five. Distribution of patients according to the WHO morphologic classification of Lupus nephritis was as follows: 6 pts showed class Ib, 6 class II, 2 class III and 13 class IV. More severe renal dysfunction, manifested by nephrotic syndrome, proteinuria with haematuria, decreased GFR and hypertension was significantly associated with proliferative lupus nephritis (combined WHO classes III and IV). Nephrotic syndrome, haematuria and decreased serum C3 with significant probability predicted proliferative lupus nephritis, while decreased GFR was of borderline predictive value.

Stimulation of malignant growth in rodents by antidepressant drugs at clinically relevant doses.

Tricyclic antidepressants, such as amitriptyline (Elavil), and the nontricyclic agent, fluoxetine (Prozac), bind to growth-regulatory intracellular histamine receptors, associated with anti-estrogen binding sites in microsomes and nuclei. The prototype anti-estrogen binding site/intracellular histamine receptor ligand, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, inhibits normal cell proliferation in vitro but stimulates tumor growth in vivo. Because of their structural similarity to N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl, we carried out studies to determine whether amitriptyline and fluoxetine stimulate tumor growth and/or development in rodents at concentrations relevant to the treatment of human depression (equivalent human dose range, approximately 100-150 mg/day for amitriptyline and approximately 20-80 mg/day for fluoxetine). All experiments were performed blinded. In studies of growth stimulation of transplantable syngeneic tumors, groups of mice were inoculated s.c. with C-3 fibrosarcoma cells or given i.v. or s.c. injections of B16f10 melanoma cells, followed 24 h later by daily i.p. injections of saline, amitriptyline, or fluoxetine. Tumor latency (fibrosarcoma), aggregate tumor weight (s.c. injected melanoma), or time to death from pulmonary metastasis (i.v. injected melanoma) was determined; drug-induced stimulation of DNA synthesis in C-3 fibrosarcoma cells in vitro was correlated with tumor growth acceleration in vivo. In a mammary carcinogenesis model, the effects of chronic saline, amitriptyline, or fluoxetine administration on the rate and frequency of development of mammary tumors in rats fed dimethylbenzanthracene (DMBA) were compared. Eight of 20 amitriptyline- or fluoxetine-treated mice developed fibrosarcoma tumors by day 5, as compared to none of 20 saline controls (P less than 0.002). Similarly, 20 of 21 DMBA-treated rats receiving the antidepressant drugs developed 33 mammary tumors by week 15 as compared to 5 tumors in 4 of 7 DMBA-treated rats receiving saline (P less than 0.001). For both models, tumor latency decreased 30-40% and, in the DMBA model, tumor frequency increased greater than 2-fold in the antidepressant-treated rats as compared to controls. Stimulation of fibrosarcoma growth in vivo correlated with a corresponding bell-shaped drug-induced increase in DNA synthesis in vitro. While the median time to death from pulmonary metastases did not differ among groups given i.v. injections of melanoma cells, a significant (P less than 0.01) stimulation of growth of s.c. injected melanoma was observed in mice receiving the antidepressants.(ABSTRACT TRUNCATED AT 400 WORDS)

The antiproliferative potency of histamine antagonists correlates with inhibition of binding of [3H]-histamine to novel intracellular receptors (HIC) in microsomal and nuclear fractions of rat liver.

Previously, we identified in rat liver microsomes, low (microM) affinity histamine receptors (HIC), associated with antiestrogen binding sites (AEBS). N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), a potent AEBS ligand, is a specific HIC antagonist. Through binding HIC, newly-formed intracellular histamine mediates, and DPPE inhibits, human platelet aggregation. We now provide evidence that histamine, mobilized from cytoplasmic stores, is a mediator of the mitogenic response to concanavalin A in mouse spleen cells. DNA synthesis and intracellular histamine levels are decreased over time by the histidine decarboxylase inhibitor, alpha-fluoromethylhistidine. For DPPE, H1 and H2 antagonists, rank order of potency to inhibit [3H]-histamine binding to HIC in rat liver microsomes correlates with antiproliferative potency. DPPE also competes for [3H]-histamine binding at low and high affinity sites in rat liver nuclei (IC50 approximately 2 microM). Thus, histamine may mediate growth through two intracellular subtypes of HIC.

[Percutaneous biopsy of the kidney in children: indications, results, and complications]. / Perkutana biopsija bubrega kod dece: indikacije, rezultati i komplikacije.

The authors present results of systematic use of percutaneous renal biopsy in children in the first three years of practice. From 1986 to 1989, 153 renal biopsies were done in 144 children and adolescents (75 males, 69 females) aged 4 months to 18 years. The most common indications were as follow: primary nephrotic syndrome, most often resistant to corticosteroid treatment (28.5%), haematuria (28.5%), secondary glomerulopathies, either with or without nephrotic syndrome (9.7% and 15.3%, respectively), and proteinuria (4.9%). 90.8% of biopsies were successful obtaining adequate tissue for light microscopic diagnosis, containing a n average number of 16 glomeruli. An overall number of 29 complications (18.9% of biopsies) in 23 patients was observed. Gross haematuria was the most common, lasting no more then one (9.8% of biopsies) or three days (2.6%) requiring blood transfusions in three cases (1.96% of biopsies). Others complications were fever (1.96%), perirenal haematoma (1.3%), and severe pain at the site of biopsy (1.3%). There were no significant differences between three annual periods with regard to the incidence of successful biopsies or complications, but more serious complications occurred during the first year of practice. In children younger than five years the rate of successful biopsies was higher and incidence of complications was lower than in older children. The authors' results correlate favourably with data published in the foreign literature.

Correlation of the antiproliferative action of diphenylmethane-derivative antiestrogen binding site ligands with antagonism of histamine binding but not of protein kinase C-mediated phosphorylation.

The nonestrogen receptor-mediated antiproliferative action of antiestrogen binding site (AEBS) ligands, including triphenylethylene antiestrogens and phenothiazines, has been linked to their ability to inhibit protein kinase C (PKC). Recent studies indicate that some diphenylmethane derivatives inhibit growth, are potent AEBS ligands, and antagonize histamine binding at an AEBS-related histamine site different from H1 and H2. Three novel diphenylmethane derivatives, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine.HCI (DPPE), 4-decanoyl-DPPE (dec-DPPE), and 4-benzylphenyl decanoate (BPD) were studied in an attempt to determine whether PKC or histamine interactions best correlate with their antiproliferative effects. Platelet aggregation and the phosphorylation of a platelet Mr 47,000 protein (p47) induced by phorbol-12-myristate-13-acetate (PMA) represent two processes mediated by PKC. DPPE inhibits PMA-induced aggregation [50% inhibitory concentration (IC50) = 31.2 +/- 2.4 (SEM) x 10(-6) M] but does not significantly inhibit either PMA-induced phosphorylation of Mr 47,000 protein (IC50 greater than 500 x 10(-6) M), or binding of [3H]phorbol dibutyrate to platelets. dec-DPPE is a more potent inhibitor of PMA-induced platelet aggregation (IC50 = 18.8 +/- 0.7 x 10(-6) M), a weak inhibitor of Mr 47,000 phosphorylation (IC50 = 80-200 x 10(-6) M), but is without effect on [3H]phorbol dibutyrate binding. BPD, which lacks the alkylaminoethoxy side chain necessary for binding to the AEBS/DPPE site, is devoid of anti-PMA effects. These results are compared to the inhibition of [3H]histamine binding in rat cortex membranes (Ki value for DPPE = 0.83 +/- 0.62 x 10(-6) M; Ki value for dec-DPPE = 6.6 +/- 3.5 x 10(-6) M; BPD is inactive) and growth inhibition of MCF-7 cells (IC50 value for DPPE = 4.5 x 10(-6) M; IC50 value for dec-DPPE = 1.5 x 10(-5) M; BPD is ineffective at all concentrations tested). Thus, while dec-DPPE is a more potent inhibitor of PKC-mediated phosphorylation, DPPE is a more potent inhibitor of histamine binding and is correspondingly more antiproliferative than dec-DPPE. The results support a relationship between antagonism of histamine binding and growth inhibition but argue against an association between the antiproliferative effects of DPPE and dec-DPPE and inhibition of PKC. The findings for DPPE suggest that platelet response to PMA, antagonized by diphenylmethane-type AEBS-ligands, may be mediated, at least in part, by mechanisms other than activation of protein kinase C-dependent phosphorylation.

Histamine and growth: interaction of antiestrogen binding site ligands with a novel histamine site that may be associated with calcium channels.

N,N-Diethyl-2-[(4-phenylmethyl)-phenoxy]ethanamine hydrochloride (DPPE) is a novel paradiphenylmethane derivative with antiproliferative and antiestrogenic properties. Like tamoxifen (TAM), DPPE binds to the microsomal antiestrogen binding site with high affinity (Kd approximately 50 nM), but, conversely, not to estrogen receptor or calmodulin. We now demonstrate that DPPE competes for [3H]histamine binding in rat cerebral cortex with an affinity (Ki = 4.5 +/- 2.6 X 10(-6) M) significantly greater than that of the H1 antagonist pyrilamine (Ki = 7.2 +/- 2.2 X 10(-5) M), despite the previous demonstration that pyrilamine is up to 1000 times more potent than DPPE in antagonizing histamine-induced contraction in canine tracheal smooth muscle. DPPE demonstrates antiproliferative activity against MCF-7 cells at concentrations between 1 X 10(-7) and 1 X 10(-5) M; the IC50 value of DPPE for growth inhibition at 7 days in this assay is 5 X 10(-6) M, a value equivalent to its Ki value for histamine binding. DPPE also competes for [3H]verapamil binding in membranes from whole rat brain with an affinity equal to that for verapamil (Kd = 4.0 +/- 1.8 X 10(-7) M); however, verapamil competes for [3H]DPPE binding in brain membranes and rat liver microsomes with an affinity markedly lower (Ki approximately 1 X 10(-4) M) than that of DPPE, suggesting allosteric interactions between the verapamil and DPPE sites. Unlike DPPE, verapamil is not antiproliferative in vitro against MCF-7 cells at concentrations up 1 X 10(-5) M, but, like DPPE, is cytotoxic at concentrations of 1 X 10(-4) M. In immature oophorectomized rats, verapamil or DPPE alone is antiuterotropic; however, verapamil shows no antagonism of exogenous estradiol on uterine growth, as opposed to DPPE which is a partial antagonist. Thus, the antiproliferative and antiestrogenic properties of DPPE either are not associated with calcium channel antagonism, or result from a qualitatively different effect on channels than verapamil. The in vitro antiproliferative effect of DPPE (7.5 X 10(-6) M) on MCF-7 cells at 72 h is significantly reversed by 10 mM L-histidine (70.2 +/- 12.6% reversal) and L-methionine (92.4 +/- 11.1% reversal), but not by L-ornithine, L-arginine, L-phenylalanine, or exogenous histamine. At lower concentrations of TAM (0.75 X 10(-6) M), where growth inhibition is estrogen-reversible, L-ornithine, but not L-histidine or L-methionine, causes significant reversal of growth inhibition (66.8 +/- 13.3%; p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)

The antiproliferative properties of tamoxifen and phenothiazines may be mediated by a unique histamine receptor (?H3) distinct from the calmodulin-binding site.

N,N-diethyl-2-[(4-phenylmethyl)-phenoxy]-ethanamine HCl (DPPE), a novel histamine antagonist (?H3), which selectively binds with high affinity to the antiestrogen-binding site (AEBS/?H3), inhibits the activity of calmodulin-dependent myosin light chain kinase (MLCK) only at concentrations greater than 1 mM, as opposed to tamoxifen (TAM), which has an IC50 = 4 microM in the same assay. This suggests that the antiestrogen-binding site is distinct from the site on calmodulin which binds TAM and phenothiazines. However, at an in vitro concentration of 1 X 10(-6) M, the antiproliferative effects of DPPE and several phenothiazines, which also compete for binding to AEBS/?H3, are about equal; this suggests that affinity for AEBS/?H3 rather than that for the calmodulin-binding site may correlate with clinically relevant antigrowth effects of these compounds.

New evidence that the antiestrogen binding site may be a novel growth-promoting histamine receptor (?H3) which mediates the antiestrogenic and antiproliferative effects of tamoxifen.

Using as a probe [3H]-DPPE (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl), a novel compound selective for the antiestrogen binding site (AEBS), new evidence is presented that this site could be a growth-promoting histamine receptor of a type not previously described (?H3). In the rat uterus, DPPE alone at a concentration of 4 mg/kg acts as an estrogen antagonist, unlike TAM alone which is a partial estrogen agonist. In the presence of exogenous estradiol, both TAM and DPPE are partial antagonists. This suggests that the "antiestrogenic" effects of tamoxifen are mediated through AEBS/?H3 while the estrogenic effects are mediated through ER.

Evidence that the antiestrogen binding site is a histamine or histamine-like receptor.

N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine X HCl (DPPE), a compound selective for the antiestrogen binding site, is structurally similar to the aminoethyl ether group of antihistamines. Our studies now reveal that H1-, but not H2-antagonists, also compete for this site in the order: DPPE = hydroxyzine = perchlorperazine greater than phenyltoloxamine greater than pyrilamine greater than diphenhydramine. The affinity of these compounds for the antiestrogen binding site correlates with their in vitro cytotoxicity against MCF-7 and EVSA-T human breast cancer cells. Tamoxifen, DPPE and hydroxyzine also bind to H1 receptors present in digitonin-solubilized rat liver microsomes, but with less affinity than pyrilamine, which is selective for this site; the ratio of H1 to antiestrogen binding sites in this preparation is 4:1. The data suggest that the antiestrogen binding site may be, in whole or in part, a receptor for histamine different from H1 and H2.