Corrected QT interval anomalies are associated with worse prognosis among patients suffering from sepsis.

BACKGROUND: Patients suffering from sepsis experience organ failure and metabolic derangements, with a negative impact on their prognosis and survival. Objective markers for dismal prognosis in this group of patients are sought. AIMS: To assess the potential role of corrected QT interval anomalies as surrogates for metabolic derangements leading to increased short and medium-term mortality in patients suffering from sepsis. METHODS: This study utilised a historic-cohort analysis of 257 septic patients admitted to internal medicine departments. Personal data, vital signs, laboratory results and electrocardiograms were collected. Patients were grouped according to QTc duration, weather mid-range (395-490 ms) or non-mid-range, and further defined as shorter (<395 ms) or longer (>490 ms). RESULTS: Mortality rates differed significantly between the mid-range QTc group and the non-mid-range groups at 14 days (23.7 vs 38.2%, respectively; P = 0.014) and at 3 months (38.5 vs 59.6%, respectively; P = 0.001). In a three-group analysis, the 14-day mortality was the highest in the longer QTc group and the lowest in the mid-range group compared with the shorter QTc group (44.4, 23.7 and 35.5%, respectively; P = 0.034), and this difference also remained at 3 months (74.1, 38.5 and 53.2%, respectively; P = 0.001). All differences remained statistically significant in a multivariate Cox regression analysis. CONCLUSIONS: QTc duration anomalies are associated with worse short- and medium-term prognosis and may act as a marker for more severe clinical sequelae.

Leptin and the post-operative inflammatory response. More insights into the correlation with the clinical course and glucocorticoid administration.

BACKGROUND: Cardiac surgery involving cardiopulmonary bypass (CPB) causes a systemic inflammatory process which can lead to multiple organ failure and postoperative morbidity. Recent animal and human studies suggested a possible involvement of leptin in the systemic inflammatory response. AIM: To characterize the response of leptin to open heart surgery (OHS) and the relationship between the time course of leptin levels and the post-operative clinical course, and to examine the effect of exogenous glucocorticoids. PATIENTS AND METHODS: Forty-seven pediatric patients, undergoing OHS for congenital heart disease were studied. Thirty-four patients (Group 1) received methylprednisolone during CPB while 13 (group 2) did not. Serial blood samples were collected perioperatively and up to 24 h after surgery, and assayed for leptin and cortisol. RESULTS: All patients' leptin levels decreased significantly during CPB (to 44-48% of baseline, p<0.001); they then increased, peaking at 12 h post-operatively. The levels of groups 1 and 2 were similar up to 8 h post-operatively; thereafter, those of group 1 were significantly higher. Recovery of leptin levels in patients with a more complicated post-operative course was comparatively slower. Cortisol levels of all patients increased significantly during CPB (p<0.001), gradually decreasing afterwards. Cortisol and leptin levels were inversely correlated in both patients' groups. CONCLUSIONS: CPB is associated with acute changes in circulating leptin levels. A complicated postoperative course is associated with lower leptin levels which are inversely correlated with cortisol levels. Leptin may participate in post-CPB inflammatory and hemodynamic responses.

Mutations in the familial Mediterranean fever gene of patients with IgA nephropathy and other forms of glomerulonephritis.

Glomerulonephritis, particularly IgA nephropathy (IgAN), seems to be more common in familial Mediterranean fever (FMF), an inherited disease caused by mutations in the MEditerranean FeVer gene (MEFV). The present study is aimed to determine, in populations not suffering from FMF, whether carriage of MEFV mutations may modify or precipitate IgAN and other forms of primary glomerulonephritis (PGN). Forty patients with biopsy proven IgAN and 40 with PGN were surveyed for the presence of the three most common MEFV mutations (M694V, V726A and E148Q), using polymerase chain reaction amplification and restriction enzyme analysis. The rate of MEFV mutations in the patients was related to the expected carrier rate in the general population of the same ethnic extraction. The effect of mutation carriage on the disease course was determined in the IgAN patient group. The frequency of MEFV mutations in IgAN or PGN was comparable to that found in ethnically adjusted general population (p = 0.1 and 0.5, respectively). Carriage of mutated MEFV was not associated with the course and severity of the disease or findings in kidney biopsy and urine analysis. In a population, mostly of Jewish extraction, MEFV mutations do not seem to predispose to the development of IgAN and other forms of PGN or affect the phenotype.

Water-soluble, dextran-linked retinal: preparation, vitamin A-like activity in rats, and effects on melanoma cells.

A new, water-soluble, polymer-linked form of retinal was synthesized and tested for its ability to support the growth of vitamin A-deficient noninbred Holtzman rats and to inhibit the proliferation of melanoma cells in culture. Retinal was conjugated to the hydrazide of carboxymethyldextran in the presence of alpha-and beta-cyclodextrins. The aqueous solutions of the product contained between 200 and 1,000 micrograms retinal/ml as opposed to the low water solubility (< 0.01 micrograms/ml) of retinal itself. The retinal-dextran complex, although barely resorbed from the gastrointestinal tract, supported the growth of rats fed a vitamin A-deficient diet when administered ip at 2.3 mumol of retinal equivalent/kg body weight. Retinal and the retinal-dextran complex exhibited differential cytotoxicity toward S91 melanoma cells and caused cell lysis at 10 and 500 microM (retinal residue), respectively. At noncytotoxic doses both free retinal and its dextran-linked derivative reduced the cell proliferation rate in a time- and dose-dependent fashion with median inhibitory doses of 1 and 4 microM (retinal residue), respectively. These data demonstrated that the water-soluble retinal-dextran complex retained certain biologic activities of retinal and was less cytotoxic.

Stimulation of melanogenesis in a human melanoma cell line by retinoids.

Retinoic acid was found to be a potent stimulant of pigmentation in human Hs939 melanoma cells. Exposure to 1 microM retinoic acid for longer than four days caused both a decrease in the rate of cell proliferation and a concomitant increase in melanogenesis. These effects of retinoic acid progressed lin-early in a time-dependent and a dose-dependent fashion such that at the end of a seven-day treatment cell growth was inhibited by approximately 65%, and both melanin content and tyrosinase activity increased more than three-fold over the control. Interpolation of the dose-response curves indicated that 3 nM retinoic acid would cause half-maximal melanogenesis stimulation. No elevation in the level of cyclic adenosine 3':5'-monophosphate could be detected in the melanoma cells following various periods of exposure to retinoic acid, and the cells were unresponsive to alpha-melanocyte-stimulating hormone. In the presence of the tyrosinase inhibitor phenylthiocarbamate, retinoic acid was capable of inhibiting cell proliferation without enhancing melanin synthesis. The tumor promoter phorbol myristate acetate did not affect either the proliferation or the differentiation of the Hs939 melanoma cells. However, the enhancement of melanogenesis by 1 microM retinoic acid was inhibited by 66% in the presence of 0.1 microM phorbol myristate acetate. The tumor promoter did not reverse the growth-inhibitory effect of retinoic acid. Phorbol, a non-tumor promoter, was effective. Other retinoids, such as 13-cis-retinoic acid, retinyl acetate, nd the trimethylmethoxyphenyl analog of retinoic acid, also inhibited the proliferation and enhanced melanin production in the Hs939 cells. In contrast, retinyl palmitate, the phenyl analog of retinoic acid, and the pyridyl analog of retinoic acid were ineffective.

Growth inhibition of murine melanoma cells by antibodies to a cell surface glycoprotein implicated in retinoic acid action.

Previous studies have shown that treatment of S91-C2 murine melanoma cells with beta-all-trans-retinoic acid (RA) results in growth inhibition, enhanced activity of sialyltransferase, and increased glycosylation of a Mr 160,000 cell surface sialoglycoprotein (gp160). None of these effects could be detected in mutant clones (e.g., S91-C154) selected from the S91-C2 cells for resistance to RA-induced growth inhibition. These findings suggest that modulation by RA of gp160 might be related causally to growth inhibition. In this study we examined the possible role of gp160 in growth regulation using specific antibodies to this glycoprotein. Metabolic labeling of S91-C2 cells with either [3H]glucosamine or [35S]methionine revealed that the cells shed into the growth medium a gp160-like glycoprotein, in addition to several other macromolecules. The gp160-like glycoprotein was isolated from concentrated conditioned medium after preparative polyacrylamide slab gel electrophoresis in the presence of sodium dodecylsulfate by excision of the corresponding protein band. Rabbits were immunized with this material and immunoblotting revealed that their sera contained antibodies that bound specifically to gp160 in extracts of untreated or RA-treated S91-C2 cells. Indirect immunofluorescence staining followed by fluorescence-activated cell sorter analysis demonstrated that the anti-gp160 antibodies bound to the surface of both untreated and RA-treated S91-C2 cells and that the treated cells bound more of the antibodies than untreated ones. In contrast, these antibodies bound to the same extent to untreated and RA-treated resistant S91-C154 cells. The growth of S91-C2 cells in the presence of anti-gp160 antibodies in semisolid medium as well as in monolayer cultures was inhibited in a dose-dependent fashion. Fifty % growth inhibition was obtained at an immunoglobulin concentration of 10 micrograms/ml. The growth of cells exposed concurrently to RA and anti-gp160 antibodies was also inhibited strongly in semisolid medium, but the antibodies caused only a small increase in the inhibitory effect of RA in monolayer cultures. No inhibition by the antibodies of either anchorage-independent growth or anchorage-dependent growth of S91-C154 cells, grown in the absence or presence of RA, was observed. These results support the suggestion that cell surface gp160 might be involved in growth regulation in the S91-C2 cells.

Differential effects of dibutyryl cyclic adenosine monophosphate and retinoic acid on the growth, differentiation, and cyclic adenosine monophosphate-binding protein of murine neuroblastoma cells.

Dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) and beta-all-trans retinoic acid (RA) have been shown separately, and in some cases in combination, to modulate the growth, differentiation, and cAMP-dependent protein kinase (PK-A) activity of various tumor cells. The effects of Bt2cAMP and RA on a cholinergic clone (S20) of C1300 mouse neuroblastoma cells were explored in the present study. Treatment of these cells with 1 mM Bt2cAMP for 3 or more days resulted in 93% inhibition of cell proliferation in monolayer cultures and in 98% inhibition of colony formation in semisolid medium (0.5% agarose). In contrast, treatment of the cells with 1 or 10 microM RA had no inhibitory effects on cell proliferation in monolayer cultures but enhanced colony formation in agarose by up to 130%. The growth of cells treated with a combination of Bt2cAMP and RA was inhibited, although less so than with Bt2cAMP alone. Cells treated with Bt2cAMP alone or Bt2cAMP and RA extended long, neurite-like, cellular processes indicative of differentiation, whereas only a few untreated or RA-treated cells produced such extensions. The amount of [3H]cAMP-binding protein increased gradually up to 2-fold during a 3-day treatment with Bt2cAMP; in contrast it decreased by nearly 2-fold during RA treatment. These changes occurred in the level of the type I regulatory subunit (RI) of PK-A as determined by photoaffinity labeling with 8-azidoadenosine cyclic 3':5'-[32P]monophosphate. The increase in RI following Bt2cAMP treatment was corroborated by DEAE-cellulose chromatography. This analysis also demonstrated that type I PK-A is the predominant kinase in the untreated S20 cells and that RI exists as a free subunit in Bt2cAMP-treated cells. The activity of PK-A decreased by about 20% following treatment with either Bt2cAMP or RA and by 45% following treatment with a combination of both agents. These results suggest that the distinct effects of Bt2cAMP and RA on the anchorage-independent growth of S20 cells may be related to their opposite effects on the level of RI.

Isolation and analysis of melanoma cell mutants resistant to the antiproliferative action of retinoic acid.

Retinoic acid inhibits both the anchorage-dependent and the anchorage-independent growth of the murine melanoma S91-C-2 cells. To explore the mechanism of these effects, several mutant cell clones resistant to retinoic acid-induced growth inhibition have been derived from the S91-C-2 cells by exposing them to the mutagen ethyl methane sulfonate and plating in soft agarose in the presence of 1 microM beta-all-trans-retinoic acid. Under such conditions, the nonmutagenized S91-C-2 cells failed to grow; however, 2 X 10(-6) of the mutagenized cells did form colonies. These colonies were isolated, expanded in culture, and recloned in agarose containing retinoic acid. Five cell clones that retained their drug-resistant phenotype after repeated subculture for 3 months, in the absence of retinoic acid, were characterized further. They were found to be 3- to greater than 1000-fold and 100- to greater than 100-fold resistant to retinoic acid-induced inhibition of anchorage-independent and anchorage-dependent growth relative to the wild-type C-2 cells, respectively. The rate of uptake of [3H]-retinoic acid by the resistant cell clones was similar to that of the sensitive C-2 cells, indicating that resistance is not the result of reduced uptake. Analysis of cytoplasmic retinoic acid-binding protein revealed that it is present in the most resistant clones in amounts that are similar to or even greater than those found in the sensitive S91-C-2 cells. These results indicate that resistance is not the result of the absence of the binding protein. The retinoic acid-resistant mutants exhibited cross-resistance to related retinoids such as 13-cis-retinoic acid and all-trans-retinol as well as to the arotinoid p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) propenyl]benzoic acid suggesting that they all share a similar mechanism of action. These resistant mutants may provide a useful system for further studies of the molecular processes through which retinoic acid exerts its antiproliferative effects.

Relationships among retinoid structure, inhibition of growth, and cellular retinoic acid-binding protein in cultured S91 melanoma cells.

S91 melanoma cells, which are sensitive to retinoic acid and contain a cellular retinoic acid-binding protein (RABP), were used in an investigation of the possible correlation between the capacities of various retinoids to inhibit cell proliferation and to bind to the RABP. Each of 27 different retinoids was evaluated for growth-inhibitory activity by exposing S91 melanoma cells to various retinoid concentrations between 1 nM and 10 microM. Subsequently, the ability of 17 of these retinoids to compete with [3H]retinoic acid for binding to RABP was determined. The results demonstrate that in addition to retinoic acid, many retinoids are capable of inhibiting the proliferation of S91 melanoma cells, although some are considerably less active. A positive correlation was found between the abilities of retinoids possessing a free carboxyl group at carbon 15 to inhibit cell proliferation and to bind to RABP. The structure-activity relationships established with the S91 cells are compared with previous reports on the biological activities of various retinoids in other systems.

Correlation of retinoic acid-enhanced sialyltransferase activity and glycosylation of specific cell surface sialoglycoproteins with growth inhibition in a murine melanoma cell system.

Retinoic acid inhibits the proliferation of the murine melanoma clone S91-C-2 cells, enhances the glycosylation of specific cell surface sialoglycoproteins, and stimulates sialytransferase activity. Mutant clones, selected from the S91-C-2 cells for resistance to the growth-inhibitory effect of retinoic acid, were used to explore whether cell surface modulation by retinoic acid is related to growth inhibition. Glycoprotein synthesis was assessed by analysis of [3H]glucosamine incorporation into glycoconjugates, and cell surface sialo- and galactoglycoproteins were analyzed after radiolabeling by the NaIO4:NaB3H4 and the neuraminidase plus galactose oxidase:NaB3H4 methods, respectively. The cells were solubilized and the labeled molecules were separated by polyacrylamide gel electrophoresis and identified by fluorography. Sialytransferase activity was measured in detergent-solubilized cells, using cytidine 5' -monophosphate-[14C]sialic acid as a sugar donor and asialofetuin as an exogenous acceptor. The results demonstrated that retinoic acid enhanced [3H]glucosamine incorporation into a Mr 160,000 glycoprotein in the S91-C-2 cells but not in any of the resistant mutant clones, while the pattern of [35S]methionine-labeled proteins was not modified in either the sensitive or the resistant clones. Radiolabeling of a Mr 160,000 sialoglycoprotein on the surface of S91-C-2 and of several retinoic acid-sensitive subclones of S91-C-2 was augmented by retinoic acid. A considerably smaller effect was observed on the labeling of Mr 160,000 sialoglycoprotein on one of the resistant clones, and no significant effect could be detected on the other resistant mutant clones. Sialytransferase activity was increased 2- to 3-fold by retinoic acid in the S91-C-2 cells and in several sensitive subclones, but not in any of the resistant mutant clones. Tetradecanoylphorbol acetate, which inhibits the proliferation of both retinoic acid-sensitive and retinoic acid-resistant cells, failed to increase either sialyltransferase activity or cell surface labeling of sialoglycoproteins. These findings suggest that the ability of retinoic acid to stimulate sialyltransferase activity and glycosylation of cell surface glycoproteins is related to the growth-inhibitory effect of this compound.

Inhibition of tumor cell colony formation in culture by a monoclonal antibody to endogenous lectins.

The presence of endogenous, galactoside-specific lectin molecules on the surface of various neoplastic cells has been demonstrated recently using monoclonal antibody (mAb) 5D7 [Raz et al., EMBO (Eur. Mol. Biol. Organ.) J., 3: 2979, 1984]. The effect of this mAb on the growth of several transformed and tumor cell lines of murine and human origin was investigated using in vitro techniques. A dose-dependent reduction (30 to 100%) in colony formation on a solid substrate or in a semisolid medium was observed when the cells were cultured in the presence of 15 to 100 micrograms of mAb 5D7 per ml of medium. Inhibition of anchorage-independent growth was more pronounced (2- to 3-fold) than inhibition of anchorage-dependent growth for most of the cells. The growth-inhibitory effects of mAb 5D7 were not the result of a cytolytic activity, for neither DNA nor protein synthesis was suppressed in semiconfluent cell cultures after 3 days of exposure to the antibody. Other mAbs that recognize cell surface components, such as chondroitin sulfate or fibronectin, failed to inhibit colony formation. These results suggest that endogenous tumor cell-surface lectin molecules may be involved in intercellular interactions or interactions between the cells and exogenous ligands; these interactions are important for growth regulation.

Biochemical and immunological characterization of K-1735P melanoma galactoside-binding lectins and their modulation by differentiation inducers.

Lectins purified by affinity chromatography on immobilized asialofetuin from extracts of mouse K-1735P melanoma cells appeared as two polypeptides [L-14.5 (Mr 14,500) and L-34 (Mr 34,000)] in one-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. However, in two-dimensional electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis) the L-14.5 polypeptide was resolved into three acidic forms of pI 4.6, 4.9, and 5.8, whereas the L-34 was resolved into two polypeptides of pI 4.9 and 5.3. Antibodies directed against galactoside-binding lectins from rat and bovine lungs, mouse 3T3 fibroblasts, and mouse UV-2237 fibrosarcoma cells reacted with the K-1735P lectins in immunoblots, and normal mouse lung extracts were found to contain cross-reactive proteins that comigrated with the two melanoma lectins. Indirect immunofluorescence staining using the above antibodies demonstrated that both L-14.5 and L-34 were expressed on the surface of viable K-1735P cells. Treatment of these cells with 1 microM beta-all-trans-retinoic acid or 1 mM N6,O2'-dibutyryl cyclic AMP for 5 days induced morphological differentiation, inhibition of anchorage-dependent and anchorage-independent growths, and a selective decrease in the L-34 lectin level. Growth inhibition by starvation for serum factors, which did not induce differentiation, had no effect on the level of L-34. These results demonstrate that the melanoma lectins are immunologically related to normal cell lectins and that the two polypeptide species are expressed on the cell surface. Further, they demonstrate that the L-34 lectin level can be modulated by agents that suppress the transformed phenotype by enhancing differentiation.

Concomitant increases in galectin-1 and its glycoconjugate ligands (carcinoembryonic antigen, lamp-1, and lamp-2) in cultured human colon carcinoma cells by sodium butyrate.

Galactoside-binding lectins (galectins) with molecular masses of about 14.5 kilodaltons (galectin-1) and 31 kilodaltons (galectin-3) have been found in a variety of normal and malignant cells and have been implicated in the regulation of cell growth, cell adhesion, and metastasis. The KM12 human colon carcinoma cell line was found to express only galectin-3. Because the levels of both galectins are developmentally regulated and can be modulated during the differentiation of several cultured tumor cell lines, we studied the ability of 11 differentiation-inducing agents to induce galectin-1 expression in the KM12 cells. Treatment of these cells with sodium butyrate, an established differentiation-inducing agent for colon carcinoma cells, resulted in the induction of galectin-1, which was detected by immunoblotting as well as by affinity chromatography. This effect was not seen with any of the 10 other differentiating agents: hexamethylene bisacetamide, dimethyl sulfoxide, dimethyl formamide, herbimycin A, mycophenolic acid, retinoic acid, difluoromethyl ornithine, dibutyryl cAMP, 8-chloro cAMP, and transforming growth factor beta 1. Galectin-1 induction by butyrate was observed in seven other human colon carcinoma cell lines. Further studies with the KM12 cells revealed that butyrate caused cell flattening, suppressed cell proliferation and colony formation in agarose, and increased the level of carcinoembryonic antigen, a marker of human colon carcinoma cell differentiation, within 48 h of treatment. The increase in galectin-1 level was dependent linearly on butyrate concentration (range, 1-4 mM). Galectin-1 mRNA expression was detected by Northern blotting as early as 6 h, and the protein was detected after 24 h of treatment initiation. The level of the constitutively expressed galectin-3 was also increased by butyrate but to a lesser extent than the level of galectin-1. Butyrate-induced galectin-1 was detected on the cell surface by immunoprecipitation from radioiodinated cell surface proteins as well as by indirect immunofluorescence labeling. Affinity-purified human galectin-1 was found to bind to purified polylactosamine-containing glycoproteins and to detergent-solubilized cellular proteins electroblotted onto nitrocellulose membranes. Affinity chromatography of [3H]glucosamine-labeled KM12 cell extracts on immobilized galectin-1 followed by immunoprecipitation from the lactose-eluted material demonstrated that lysosome-associated membrane glycoprotein-1, carcinoembryonic antigen, and nonspecific cross-reacting antigen are the major galectin-1-binding proteins in these cells. These results indicate that galectin-1 expression may be associated with the differentiation of KM12 cells and that several glycoproteins shown to be important in colon carcinoma adhesion and metastasis are capable of functioning as its endogenous ligands.

Overexpression of retinoic acid receptor beta in head and neck squamous cell carcinoma cells increases their sensitivity to retinoid-induced suppression of squamous differentiation by retinoids.

Nuclear retinoic acid receptor beta(RARbeta) expression is suppressed in many head and neck squamous cell carcinomas (HNSCCs), and an inverse relationship was found between squamous differentiation and RARbeta expression in such cells. To investigate the role of RARbeta in HNSCC growth and differentiation, we transfected a retroviral RARbeta2 expression vector (LNSbeta) into HNSCC SqCC/Y1 cells, which do not express endogenous RARbeta but do express RARalpha, RARgamma, and retinoid X receptors. Transfected clones expressing RARbeta2 mRNA and protein exhibited enhanced sensitivity to the suppressive effects of all-trans-retinoic acid (ATRA) on squamous differentiation compared with cells transfected with the LNSX vector only; transglutaminase type I level was suppressed after a 3-day treatment with 10(-10) M ATRA in four of five LNSbeta clones, whereas it was not suppressed in LNSX cells even by 10(-6) M ATRA. Similarly, cytokeratin 1 mRNA level was more suppressed in ATRA-treated LNSbeta clones than it was in LNSX cells. This effect was independent of transrepression of activator protein-1. None of the LNSbeta-transfected clones showed an increased growth inhibition by ATRA, 9-cis-retinoic acid, or the synthetic retinoid 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthale necarboxylic acid. These findings suggest that, in SqCC/Y1 cells, RARbeta mediates suppression of squamous differentiation by ATRA without enhancing its growth-inhibitory effects.

Modulation of growth and epidermal growth factor receptor activity by retinoic acid in human glioma cells.

The growth-inhibitory activity of beta-all-trans-retinoic acid (RA) was examined on seven cultured human gliomas and cells derived from one normal brain. Response in monolayer cultures was heterogenous: three cell lines were completely resistant whereas five cell lines were growth inhibited with 50% inhibitory dose ranging from greater than 10(-5) to 1 x 10(-8) M. Two glioma cell lines capable of forming colonies in soft agar exhibited dose-dependent sensitivity to RA-induced growth inhibition, whereas another cell line was not affected by RA under either growth condition. Cell cycle analysis of the glial-derived cells has shown that the RA-sensitive cells accumulated in the G0-G1 phase. The cell surface expression of epidermal growth factor (EGF) receptors displayed by the various cells was either slightly increased or not affected by RA. In addition, the affinity of binding was slightly decreased in some sensitive cells. The activity of EGF receptor as assessed by immunocomplex-kinase assays revealed a dose-dependent decrease in autophosphorylation activity that appeared to correlate with the growth inhibition. The decrease in phosphokinase activity represented a dose-dependent inhibition of phosphorylation on tyrosine residues on EGF receptor as well as several other substrates. Furthermore, the autophosphorylation of either RA-treated or untreated EGF receptors occurred on similar amino acid residues. These results demonstrate that RA exhibits a heterogeneous growth-inhibitory activity against human glioma cells and suggest that the effects of RA may be mediated, at least in part, by modulation of EGF receptor phosphotyrosine kinase activity.

Enhanced efficacy of combinations of retinoic acid- and retinoid X receptor-selective retinoids and alpha-interferon in inhibition of cervical carcinoma cell proliferation.

Retinoic acid receptors and retinoid X receptors form heterodimers, bind to retinoic acid response elements, and transactivate the transcription of retinoid-responsive genes. Two synthetic retinoids [4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid (TTAB) and 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-naphthale n ecarboxylic acid (TTNN)], which preferentially bind retinoic acid receptors, inhibited the proliferation of cervical carcinoma ME180 cells by 50% at 0.2 nM and 0.2 microM, respectively. In contrast, two other retinoids [2-(4-carboxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-1,3-dithiane (SR11203) and 4-(2-methyl-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)propenyl)benzoic acid (SR11217)], which preferentially bind retinoic X receptors, inhibited growth by only 12 and 18% at 1 microM, respectively. The combination of suboptimal concentrations of TTAB (0.1 nM) or TTNN (10 nM) with each of the retinoic X receptor-selective retinoids at 1 microM showed more than additive effects on cell proliferation, especially with SR11217. Further increases in proliferation inhibition were observed when IFN-alpha (100 units/ml) was added to these retinoid combinations. Activation of transcription of a reporter gene linked 3' to the retinoic acid receptor beta retinoic acid response element in transiently transfected cells also exhibited additive effects when the cells were treated with combinations of TTAB or TTNN with SR11217. This additive activation of transcription may be the reason why the combination of retinoids is more effective than each retinoid alone. The results also suggest that the use of combinations of retinoids and IFN-alpha may lead to enhanced antitumor effects.

Carcinoembryonic antigen and other glycoconjugates act as ligands for galectin-3 in human colon carcinoma cells.

Galectin-1 and galectin-3, galactoside-binding lectins with molecular weights of M(r) 14,500 and 31,000, respectively, are expressed in normal and malignant cells and have been implicated in regulation of cell growth, adhesion, and metastasis. We analyzed the expression of galectins in 21 cultured human colon carcinoma cell lines by immunoblotting. Galectin-1 was detected in only 7, whereas galectin-3 was found in 20 of the cell lines. KM12 cells, which express only galectin-3, were used to isolate this lectin by affinity chromatography, and the purified lectin was used to identify complementary glycoconjugates by blotting. Galectin-3 was shown to bind to human laminin, carcinoembryonic antigen, and lysosome-associated membrane glycoproteins, which are involved in cell adhesion. Galectin-3 was localized on the KM12 cell surface and colocalized with carcinoembryonic antigen. Several endogenous glycoproteins and cell surface proteins of molecular weights in the range M(r) 58,000 to > 200,000, including carcinoembryonic antigen and lysosome-associated membrane glycoproteins, were identified as galectin-3 ligands by coimmunoprecipitation with and affinity chromatography on immobilized galectin-3. These data demonstrate that galectin-3 interacts with several adhesion molecules and suggest that this lectin may have a role in human colon carcinoma cell adhesion.

Inhibition by retinoic acid of type IV collagenolysis and invasion through reconstituted basement membrane by metastatic rat mammary adenocarcinoma cells.

The activity of type IV collagenase, which enables tumor cells to degrade collagen type IV found in the subendothelial basement membrane, has been correlated with the metastatic potential in several tumor types, including the rat 13762NF mammary adenocarcinoma cell line and its clones. In this study, we examined whether all-trans-retinoic acid (all-trans-RA) and other retinoids, which exhibit antitumor activity in vitro and in vivo, affect the collagenolytic activity of metastatic rat 13762NF mammary adenocarcinoma cells. Cells of the highly metastatic lung-colonizing clone MTF7.T35.3, derived from the 13762NF cell line, were treated for 3 days with 0.1, 1, or 10 microM all-trans-RA, harvested, and seeded on [3H]proline-labeled extracellular matrix deposited by cultured rat lung endothelial cells or on a film of purified [3H]proline-labeled type IV collagen. The amount of radioactivity released into the medium during the subsequent 24 to 72 h was measured, and it was found that all-trans-RA treatment inhibited degradation of extracellular matrix and type IV collagen by 50 to 60%. This effect was observed whether the cells had been treated with all-trans-RA in serum-free medium or in medium supplemented with heat-inactivated or acid-treated fetal bovine serum. The growth of the cells was not inhibited under these conditions, except after treatment with 10 microM all-trans-RA in serum-free medium. The reduction in collagenolytic activity was observed in viable cells as well as in conditioned medium. A 24-h exposure of cells to all-trans-RA was sufficient to cause a 30% decrease in the collagenolytic activity, and this inhibitory effect was reversible. The direct addition of all-trans-RA to conditioned medium had no effect on secreted collagenase activity. The apparent molecular weights of the collagenolytic enzymes were determined by electrophoresis of cell extracts and concentrated conditioned medium in type IV collagen-embedded polyacrylamide gels followed by renaturation and activation of the enzymes within the gels. Two major type IV collagenolytic metalloproteinases exhibiting molecular weights of 64,000 and 88,000, respectively, were detected by this method. These two enzymes were also found to have specificity for gelatin. The Mr 64,000 enzyme could be extracted from viable cells (presumably from the cell membrane) by 2% 1-butanol. Treatment with all-trans-RA decreased the level of these enzymes in the cellular, cell membrane, and conditioned medium compartments.(ABSTRACT TRUNCATED AT 400 WORDS)

Retinoic acid inhibition of human melanoma cell invasion through a reconstituted basement membrane and its relation to decreases in the expression of proteolytic enzymes and motility factor receptor.

Treatment of four A375 human melanoma sublines (A375, A375P, A375P-5, A375M), exhibiting distinct metastatic potentials in vivo, with beta-all-trans-retinoic acid in vitro caused a dose- and time-dependent inhibition of the ability of these cells to penetrate Matrigel-coated filters using a reconstituted basement membrane invasion assay. The possible mechanisms of action responsible for the antiinvasive effect were further investigated, and the data showed that compared with untreated cells the retinoic acid-treated cells: (a) secreted lower levels of collagenolytic enzymes, as demonstrated by a decreased ability of the cells to degrade [3H]proline-labeled type IV collagen substrate and by a reduction in the activity of a secreted Mr 64,000 collagenolytic enzyme detected in type IV collagen-containing polyacrylamide gels; (b) expressed lower levels of the human type IV collagenase mRNA (except in the A375P cells), as detected by Northern blot analysis; (c) exhibited decreased levels of tissue plasminogen activator activity, as demonstrated by a chromogenic assay; (d) were 10-40% less adhesive to a reconstituted basement membrane matrix, as determined by a 60-min Na2(51)CrO4-labeled cell attachment assay; (e) exhibited an increase in the high affinity metastasis-associated cell surface laminin receptor, as determined by flow cytometry after binding of fluorescently labeled laminin receptor antibody; and (f) expressed decreased amounts of gp78, a cell surface receptor for motility factor, demonstrated by immunoblotting and immunofluorescence. Collectively, these data suggest that retinoic acid inhibits tumor cell invasion through a basement membrane-like matrix by suppressing matrix degradation and by altering cell surface receptors.

Implication of retinoic acid receptor gamma in squamous differentiation and response to retinoic acid in head and neck SqCC/Y1 squamous carcinoma cells.

Nuclear retinoic acid receptors are considered to be the mediators of most of the effects of retinoic acid (RA) on gene expression. To explore the role of RA receptor gamma (RARgamma) in the growth and differentiation of SqCC/Y1 head and neck squamous carcinoma cells, they were transfected with RARgamma sense and antisense expression vectors and stable clones in which RARgamma expression was either increased or blocked were isolated. The growth inhibitory effect of RA in monolayer culture was enhanced in the sense transfectants and decreased in the antisense ones. The ability to form colonies in semisolid medium was abolished by RA in the sense transfectants, while the antisense transfected clones exhibited heterogeneous responses. The expression the squamous differentiation markers cytokeratin K1 transglutaminase type I, and involucrin was increased in the absence of exogenous retinoid in a sense transfected clone and decreased in an antisense transfected clone. RA suppressed squamous differentiation in both types of transfectant. The expression of epidermal growth factor receptor (EGFR) was higher in the antisense and lower in the sense transfectant than in the parental cells and RA decreased EGFR mRNA level in the parental and the sense transfectant but not in the antisense transfectant. In addition activator protein-1 (AP-1) binding activity was decreased by the RA treatment in the sense clones, but not in the antisense ones. These results suggest that RARgamma mediates the effects of RA on the cell growth both in monolayer culture and in semisolid medium possibly through AP-1 suppression.