Use of quantitative polymerase chain reaction to study retinoid receptor expression.
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Animals↗
Biomedical subjects
G Vidali
Publications and source records attributed to G Vidali.
Micellar electrokinetic capillary chromatography of three natural vitamin A derivatives.
Micellar electrokinetic chromatography (MEKC) using bile salts has been employed to separate retinoids differing in structure and charge; bile salts in MEKC allows the separation of liposoluble molecules but, to the best of our knowledge, there are only few data on the above-mentioned technique for the separation of highly hydrophobic compounds. The three natural vitamin A derivatives, retinal, retinol and retinoic acid, were successfully separated by MEKC using sodium cholate within a relatively short time (ca. 25 min), whereas the separation of these compounds was not successful using sodium dodecyl sulfate or sodium deoxycholate. Several parameters (pH and organic modifiers, in addition to bile salts concentration) have been tested to provide a system that can be extended to synthetic retinoids, which are often used in treating several diseases, including cancer prevention and therapy.
Buffers↗
Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines.
Several laboratories have described estrogen receptor mRNA variants created by skipping internal exons. Some of the putative proteins encoded for by these variants have been functionally characterized by transfection analyses. The variant lacking exon 5 would lead, if translated, to a truncated receptor which shows dominant positive transactivation activity in the absence of hormone. It has been postulated that the variant could account for anti-estrogen resistant tumor growth and for expression of the progesterone receptor in estrogen negative tumors. In order to understand the possible role this and other variants may have in the tumorigenesis of mammary tissue we have carried out a thorough analysis of variants expressed in a tumor cell line (MCF-7), in a tumor sample and in a sample of normal breast tissue derived from mammary reduction surgery. We performed rt-PCR analyses followed by hybridization with exon specific oligonucleotide probes. By these means we have detected nine different variants co-expressed in MCF-7 cells and at least the major variants were equally expressed in normal and neoplastic breast tissue. The same is true for the variant lacking exon 5 which, however, resulted to be a variant of low expression in the three samples analyzed. Variant formation appeared to be restricted to the estrogen receptor messenger since several other members of the superfamily of nuclear receptors did not show variant formation. We also have analyzed the effect of the most abundantly expressed variant, the exon 4 lacking variant, on normal estrogen receptor function, on the growth and on the response to estradiol and to tamoxifen of MCF-7 cells. Although over-expressed at high levels this variant has, if any, only marginal effects on the expression of endogenous estrogen regulated genes and on growth and response to the hormone and its antagonist. Although the lack of function of this variant cannot be extrapolated to other variants, their involvement in tumor formation appears rather unlikely since they are also expressed in normal tissue and the single variant is expressed in addition to many others, some of which might have opposing effects. Variant formation is, however, specific for the estrogen receptor and apparently regulated with tissue specificity as our expression analysis in normal mouse tissues shows. Therefore the variants probably have a physiological significance yet to be discovered.
Adenocarcinoma↗
Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes.
Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4(+) and CD8(+) T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10(-3)-10(-12)M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G1 phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8(+) than on CD4(+) T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.
Adult↗
Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissues and in MCF-7 cells.
Mammary cancers often develop into a hormone-independent and antagonist-resistant growth phase. The molecular mechanisms of this transition are not clear. Recently, it has been proposed that estrogen receptor variants derived from alternative splicing might lead to dominant positive transcription factors acting on estrogen response elements, even in the absence of the hormone. We show here the comprehensive analysis of expression of estrogen receptor variants lacking internal exons in the estrogen receptor-positive mammary carcinoma cell line MCF-7, in a tumor sample, and in healthy breast tissue taken from reduction surgery. Variants are identified by reverse transcription PCR and hybridization to exon-specific oligonucleotide probes. In MCF-7 cells we detected 10 variants including 5 that have not been described before. Skipping one, two, or three exons occurs. The major variants detected in the cell line are also present in normal and neoplastic tissues. Quantitative variations allow no conclusions of a potential involvement of the variants in neoplastic processes. Rather, the variants appear to be present normally and thus might have a physiological role. Given the expression of the variants in normal tissue, and given the expression of potentially dominant positive variants in conjunction with potentially dominant negative ones, we suggest that these variants do not account for hormone antagonist resistance.
Amino Acid Sequence↗
A retinoic acid resistant HL-60 cell clone sensitive to N-(4-hydroxyphenyl) retinamide-mediated clonal growth inhibition.
Among the Retinoic Acid (RA) derivatives, retinamides, and in particular N-(4-hydroxyphenyl) retinamide (4-HPR), are currently being investigated in selected cases of cancer chemoprevention. The cellular target range, however, seems to be limited, as cells of hemopoietic origin are virtually incapable of terminal differentiation upon addition of the compound. We have reconsidered the effect of 4-HPR on HL-60 cells by taking advantage of a mutant clone, generated in our laboratory, unresponsive to RA but highly responsive to dimethylsulfoxide (DMSO). We show here that this clone, upon addition of 4-HPR, although unable of undergoing full differentiation, shows considerable reduction of clonal growth. Moreover, the combination of 4-HPR and RA resulted in a much greater effect than the administration of 4-HPR alone. We suggest that 4-HPR and RA, at least in terms of mediating growth inhibition, may follow different metabolic pathways.
Animals↗
Efficient one-tube RT-PCR amplification of rare transcripts using short sequence-specific reverse transcription primers.
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Base Sequence↗
Estradiol inhibits growth of hormone-nonresponsive PC3 human prostate cancer cells.
Significant inhibition of proliferative activity in PC3 human prostate cancer cells by estradiol is reported, accompanied by experimental evidence for a specific estrogen receptor (ER). Radioligand-binding assays revealed the presence of high affinity sites of estrogen binding in the nuclear compartment of PC3 cells. In addition, using a reverse transcriptase-polymerase chain reaction system, we obtained evidence of either normal or a variant ER mRNA; the latter, which lacks the entire exon 4, is coexpressed with normal ER mRNA and has been recently characterized in our laboratories. The likelihood that the inhibitory effect exerted by estradiol could be mediated by an increase of transforming growth factor beta (TGF beta) production was also investigated. Use of monoclonal antibodies against TGF beta 1 produced a 3-fold increase of growth rate in PC3 cells; this clearly speaks for high levels of endogenous TGF beta 1. This effect was almost completely abolished after addition of 100 nM estradiol. However, we failed to demonstrate any increase of TGF beta 1 mRNA after estradiol administration using Northern blot analysis. Further studies are needed to ascertain whether the estradiol-induced growth inhibition of PC3 cells is either mediated by other TGF beta species or exerted via alternative mechanism(s).
Blotting, Northern↗
An improved RT-PCR protocol for the quantitation of human retinoic acid receptor RNA.
A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) system has been developed to calculate the level of expression of human retinoic acid receptors (hRAR) alpha, beta, and gamma. Starting from a single cDNA preparation, the system allows the measurement of the number of molecules of each mRNA receptor. This is made possible by a synthetic internal standard mRNA which is added in known concentrations at the beginning of the reaction. The system is tested in a rhabdomyosarcoma cell line (A-673) where we have measured the upregulation of beta and gamma receptor mRNAs following treatment with retinoic acid.
Base Sequence↗
Estrogen receptor variant messenger RNA lacking exon 4 in estrogen-responsive human breast cancer cell lines.
We have identified a messenger RNA coding for a variant estrogen receptor jointly expressed with the normal mRNA in the estrogen receptor-positive, -responsive mammary carcinoma cell lines MCF-7 and ZR 75-1 by means of reverse transcription polymerase chain reaction. This variant mRNA was not observed in estrogen receptor-negative, -unresponsive MDA-MB 231 cells. Partial sequence analysis of the variant complementary DNA revealed identity to sequences of the estrogen receptor exons 3, 5, and 6, but the absence of the entire exon 4. We suggest that this variant receptor messenger is created by alternative splicing. The variant protein is expected to lack most of the hinge domain and part of the hormone binding domain, and it might have a cellular distribution and estrogen-binding affinity different from that of the normal receptor protein.
Amino Acid Sequence↗
Quantitative analysis of mitotic and early-G1 cells using monoclonal antibodies against the AF-2 protein.
We have recently described a novel protein (AF-2), conserved between fission yeast and man, and we have shown by flow cytometry (FCM) that AF-2 is highly accessible to specific monoclonal antibodies (MoAbs) in mitotic and postmitotic early-G1 phase cells. The aim of the present study was to optimize the FCM methodology using MoAbs against AF-2 and to show that the evaluation of the mitotic cells, using different cell lines, was quantitative and reproducible. We found that a method based on fixation with ethanol, instead of formalin, resulted in improved DNA histogram coefficients of variation and implemented separation of early-G1 cells from late-G1 cells. In addition, by eliminating several cell permeabilization and protein salt extraction steps, the method became straightforward, conserved a clear-cut separation of the green fluorescence of M- with respect to G2-phase cells, and did not significantly affect cellular integrity. The coefficient of correlation among the mitotic index values evaluated by this FCM method using MoAbs against AF-2 and by microscopic visual counting was R = 0.94. When the FCM/AF-2 method was tested against an independent FCM method, which allows clear separation of M- and G2-phase cells according to 90 degrees scattering, we found R = 0.93. We conclude that MoAbs against the AF-2 protein may be used in FCM for quantitative analysis and for isolation of M-phase cells, providing as well, the identification of the early-G1 cell subcompartment. The method may, in addition, be useful for the simultaneous detection of cytoplasmic cytokeratin and nuclear AF-2 antigen.
Antibodies, Monoclonal↗
An essential yeast gene encoding a TTAGGG repeat-binding protein.
A yeast gene encoding a DNA-binding protein that recognizes the telomeric repeat sequence TTAGGG found in multicellular eukaryotes was identified by screening a lambda gt11 expression library with a radiolabeled TTAGGG multimer. This gene, which we refer to as TBF1 (TTAGGG repeat-binding factor 1), encodes a polypeptide with a predicted molecular mass of 63 kDa. The TBF1 protein, produced in vitro by transcription and translation of the cloned gene, binds to (TTAGGG)n probes and to a yeast telomeric junction sequence that contains two copies of the sequence TTAGGG separated by 5 bp. TBF1 appears to be identical to a previously described yeast TTAGGG-repeat binding activity called TBF alpha. TBF1 produced in vitro yields protein-DNA complexes with (TTAGGG)n probes that have mobilities on native polyacrylamide gels identical to those produced by partially purified TBF alpha from yeast cells. Furthermore, when extracts are prepared from a strain containing a TBF1 gene with an antigen tag, we find that the antigen copurifies with the predominant (TTAGGG)n-binding activity in the extracts. The DNA sequence of TBF1 was determined. The predicted protein sequence suggests that TBF1 may contain a nucleotide-binding domain, but no significant similarities to any other known proteins were identified, nor was an obvious DNA-binding motif apparent. Diploid cells heterozygous for a tbf1::URA3 insertion mutation are viable but upon sporulation give rise to tetrads with only two viable spores, both of which are Ura-, indicating that the TBF1 gene is essential for growth. Possible functions of TBF1 (TFB alpha) are discussed in light of these new results.
Amino Acid Sequence↗
Regulation of plasma retinol binding protein secretion in human HepG2 cells.
Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During vitamin A deficiency, RBP secretion is specifically inhibited while its rate of biosynthesis is unaffected. As a consequence, RBP, as apoprotein, accumulates inside the endoplasmic reticulum (ER) of the hepatocyte, and a new elevated steady-state concentration is reached. We have studied the role of degradation on the regulation of RBP metabolism in retinol deficient HepG2 cells and determined the intracellular site where RBP degradation takes place. Pulse-chase experiments show that RBP half-life is ca.9 h in retinol-depleted cells. RBP degradation is slow and is insensitive to the treatment with NH4Cl, which inactivates lysosomal proteases and to the drug brefeldin A, which prevents protein export from the ER. The data obtained suggest that RBP degradation occurs, at least in part, in a pre-Golgi compartment. 2-Mercaptoethanol, at millimolar concentration, induces RBP secretion, suggesting a possible role for sulfhydryl-mediated apo-RBP retention by resident ER proteins.
Carcinoma, Hepatocellular↗
Chromatin structure, DNA methylation, and gene expression at sites of viral integration in human fibroblasts. Implications for chromosomal fragility.
We analyzed the structural and functional properties of a chromosomal region in which a recombinant hybrid virus adenovirus 5/SV40 preferentially integrates. Our results demonstrated that the structure of the cellular targets for DNA and RNA viruses is very similar and that the cellular sequence flanking the integrated virus possesses, simultaneously, all the features postulated to be the molecular basis for chromosomal fragility.
Adenoviridae↗
Post-transcriptional control of H3 histone variants synthesis.
In cultured HeLa cells the rates of H3.1 and H3.2 synthesis measured by pulse labeling experiments reflect the steady state content of the two histone variants. This pattern, however, is largely modified when histone translation is carried out in vitro on RNA isolated from the same cell line. In vivo, H3.1 and H3.2 are synthesized approximately at the same rates while the product of H3 mRNA translation in vitro is mostly represented by H3.1 histones. Factors which have so far been invoked for the control of histone messenger RNA stability and translation efficiency are not sufficient to explain our data which in addition indicate that histone H3.1 and H3.2 have different roles in the organization of the genetic material.
Autoradiography↗
Cell cycle dependent alterations of chromatin structure in situ as revealed by the accessibility of the nuclear protein AF-2 to monoclonal antibodies.
We have recently described a novel nuclear antigen, AF-2, which is related to cell cycle dependent alterations of chromatin structure. We show by two parameter flow cytometry on a cell by cell basis that the antigen is accessible to specific monoclonal antibodies only in mitotic and postmitotic early G1-phase cells. The evaluation of nuclease susceptibility and AF-2 antigen accessibility reveals different subcompartments of the G1-phase of the cell cycle with distinct chromatin conformations. Digestion with DNase I seems to alter the chromatin structure according to concentration and this is reflected by an increase of the antigen accessibility. Chromatin in the more condensed early G1-phase is specifically digested by lower concentrations of the enzyme than chromatin in later stages of interphase. Chromatin from cells in the late-G1, S-, and G2-phases shows a higher relative resistance to DNase I and a reduced accessibility of the AF-2 antigen to monoclonal antibodies. Nuclease S1 has a similar effect on chromatin topology, as revealed by the reaction with anti-AF-2 antibodies, without digestion of detectable amounts of DNA. The antigen becomes available to the antibodies in almost all cells by digestion with high concentrations of DNase I or Nuclease S1.
Antibodies, Monoclonal↗
A novel protein related to cell cycle-dependent alterations of chromatin structure.
We have detected a novel nuclear antigen, AF-2, which appears to be involved in cell cycle-dependent alterations of chromatin structure. Specific monoclonal antibodies detect the antigen spread over the whole cell during mitosis and in islet-like structures in the nuclei of a subpopulation of cells in interphase. Upon nucleolytic digestion of fixed cells, the antigen becomes available to the antibodies in all cells, indicating that AF-2 antigen is present during the whole cell cycle but differentially accessible. Digestion with the single strand specific S1 nuclease reveals that the alteration of chromatin structure induced by the introduction of nicks into the DNA rather than the digestion of DNA bound to the immunogenic epitope accounts for the change in accessibility of AF-2 antigen in interphase nuclei. The epitope recognized by the antibody in human cells is present in two polypeptides of 65 and 36 kDa, respectively, which are tightly bound to chromatin and cross-linkable to the nuclear matrix. The proteins also occur in the midbody during cytokinesis. The immunogenic epitope is conserved between man and fission yeast.
Antibodies, Monoclonal↗
Histone acetylation: recent approaches to a basic mechanism of genome organization.
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Acetylation↗