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J Kokontis

Publications and source records attributed to J Kokontis.

15 recordsLinked to original sources
PubMed1996

p21 Disrupts the interaction between cdk2 and the E2F-p130 complex.

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G1 phase is associated with a cyclin-dependent binding of the cyclin-dependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32 degrees C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculated that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.

Animals↗
PubMed1994

Increased androgen receptor activity and altered c-myc expression in prostate cancer cells after long-term androgen deprivation.

Proliferation of LNCaP 104-S cells, a clonal subline of the human prostate cancer cell line, was very slow in androgen-depleted medium but increased 10-13-fold in the presence of 0.1 nM of a synthetic androgen, R1881. This induction of proliferation was diminished at higher concentrations of R1881, indicating the biphasic nature of the androgen effect. After 20-30 passages in androgen-depleted medium, these cells progressed to 104-I cells, which exhibited much lower proliferative sensitivity to 0.1 nM R1881. After another 20-30 passages, LNCaP 104-I cells gave rise to 104-R cells, which proliferated rapidly without additional androgen. Proliferation of 104-R cells was induced 2-fold by 0.01 nM R1881 but was repressed by 0.1 nM R1881 and above. Thus, androgen induction and repression of proliferation could be seen at lower concentrations of androgen as the cells progressed. During the transition of 104-S cells to 104-R cells, the androgen receptor mRNA level increased 2.5-fold whereas the androgen receptor protein level increased 15-fold in the absence of androgen. Androgen receptor transcriptional activity, measured by androgen induction of prostate-specific antigen mRNA and chloramphenicol acetyltransferase activity in transfected cells, increased up to 20-fold during the progression. LNCaP cells, therefore, appear to be able to adapt to reduced androgen availability by increasing their sensitivity to androgen, raising questions concerning the therapeutic strategies used against prostate cancer. Androgen induction of c-myc expression in 104-R cells occurred at a 10-fold lower concentration (0.01 nM) than in 104-S cells (0.1 nM). In all stages, cell proliferation and c-myc expression were repressed by androgen at a high concentration (20 nM), but the repression of cell proliferation was blocked by retroviral overexpression of c-myc.

Androgens↗
PubMed1993

The use of a DNA-binding domain replacement method for the detection of a potential TR3 orphan receptor response element in the mouse mammary tumor virus long terminal repeat.

TR3 orphan receptor is a human homologue of the mouse nur77, N10 and rat NGFI-B, TIS1 genes which may represent an early response gene involved in the control of cell proliferation. We have studied potential target genes for TR3 orphan receptor using the DNA-binding domain replacement method. We found that mouse mammary tumor virus long terminal repeat-linked chloramphenicol acetyltransferase expression can be activated in transfected cells by a chimeric androgen receptor/TR3 orphan receptor/androgen receptor construct (AR/TR3/AR) in the presence of androgen. By deletion analysis, a region with 20 nucleotides in length between positions -1178 and -1159 of the mouse mammary tumor virus long terminal repeat was confirmed as a potential TR3 orphan receptor response element. These results suggest that feasibility of using the DNA-binding domain replacement method to detect target sequences of orphan receptors.

Androgens↗
PubMed1991

Autoregulation of androgen receptor expression in rodent prostate: immunohistochemical and in situ hybridization analysis.

Autoregulation of androgen receptor mRNA and protein was investigated by immunohistochemical and in situ hybridization techniques. In both mouse and rat prostate, the epithelial cell nuclei were stained with the monoclonal or polyclonal antibodies raised against human androgen receptor. It was observed that 3 days after castration, nuclear staining of the epithelium was greatly reduced, while androgen treatment restored the staining intensity to a normal level. In situ hybridization using an androgen receptor cDNA fragment as probe demonstrated that the change in androgen receptor mRNA level correlated with the change in antibody staining intensity. These data suggested an up-regulation of androgen receptor expression by androgen.

Animals↗
PubMed1991

Expression and function of normal and LNCaP androgen receptors in androgen-insensitive human prostatic cancer cells. Altered hormone and antihormone specificity in gene transactivation.

The gene for the androgen receptor (AR) in the androgen-sensitive human prostate cancer cell line LNCaP has a single-base mutation that produces a threonine to alanine change in the androgen-binding domain. Androgen-insensitive prostatic cancer (PC-3) cells were cotransfected with an expression vector encoding normal, LNCaP, or chimeric normal/LNCaP AR and a vector carrying a chloramphenicol acetyltransferase (CAT) reporter gene linked to the mouse mammary tumor virus promoter. CAT activity was specifically induced by androgens in PC-3 cells expressing normal AR. In PC-3 cells expressing LNCaP AR, however, CAT activity was also induced by progestins and the antiandrogen hydroxyflutamide, which had little activity in cells expressing normal AR. Steroid-binding competition assays using in vitro synthesized ARs showed that LNCaP AR had a higher affinity than normal AR for progestins, 17 beta-estradiol, and hydroxyflutamide. The antiprogestin and antiglucocorticoid RU 38486 induced CAT activity in PC-3 cells expressing normal AR but not LNCaP AR. These studies indicate that AR mutations may be very important in determining the appropriate method of treatment with steroid hormones or their antagonists.

Androgen Antagonists↗
PubMed1991

Transcriptional activation by TR3 receptor, a member of the steroid receptor superfamily.

TR3 receptor is a human homolog of mouse Nurr77 and N10 protein and the rat NGFI-B protein. A cDNA encoding a chimeric nuclear receptor composed of the N-terminal domain and C-terminal putative ligand-binding domain of the orphan receptor TR3 receptor and the DNA-binding domain of the androgen receptor was constructed. The chimeric receptor, called TR3/AR/TR3 receptor, when expressed in COS-1 monkey kidney cells or PC-3 human prostate tumor cells, cotransfected with an ARE-containing mouse mammary tumor virus long terminal repeat-linked reporter gene encoding chloramphenicol acetyltransferase (CAT), activated CAT expression in the absence of any added factor. The activation was dependent on the amount of expression vector transfected and appeared to be independent of the concentration of serum supplement. Intact TR3 receptor was not active in this system. A TR3/AR/TR3 receptor protein truncated in the putative ligand-binding domain also induced CAT activity. TR3 receptor appears to be a transcriptional factor that activates transcription independently of ligand or binds an endogenous ligand present constitutively in cultured cells.

Base Sequence↗
PubMed1989

Molecular cloning of new human TR2 receptors: a class of steroid receptor with multiple ligand-binding domains.

Previously we isolated a new group of cDNA clones from human testis cDNA libraries which might code for new steroid receptors. The cDNA and predicted amino acid sequences of two of these receptors, named TR2-5 and TR2-7 receptors, were determined. We report here the nucleotide and deduced amino acid structures of two other receptors that we named TR2-9 and TR2-11 receptors. The calculated MW of TR2-5 receptor, TR2-7 receptor, TR2-9 receptor and TR2-11 receptor are 52,982, 20,528, 50,849 and 67,223 respectively, which match well with the apparent MW of in vitro translated products. The 26 amino acids involved in the formation of "Zn-fingers" are conserved. The ligand-binding domain of TR2-9 receptor is 16 amino acids shorter and has 3 different amino acids compared with TR2-5 receptor. The TR2-11 receptor has a ligand-binding domain which is longer and quite different compared with the other TR2 receptors. The multiple ligand-binding domains of TR2 receptor could be the products of different genes or may be due to RNA splicing errors. So far, we have failed to find binding activity with any known steroid hormone; this promotes the possibility that an unidentified steroid hormone may be involved.

Amino Acid Sequence↗
PubMed1989

Androgen receptors: structures, mutations, antibodies and cellular dynamics.

An overview of recent studies from this and other laboratories on the structures and intracellular dynamics of androgen receptors is presented. Human and rat androgen receptors are unique in that, aside from their DNA and androgen binding domains, they have amino terminal regions rich in oligo- and poly(amino acids) motifs as in some regulatory and homeotic genes. Point mutations that cause sequence changes or deletion of regions of androgen receptors appear to be responsible for some cases of androgen-insensitivity. Monoclonal antibodies produced against specific regions of the androgen receptor bind to androgen receptors but not other major steroid receptors. Androgen receptors in the human and rat prostate, and monkey seminal vesicle were localized to the nucleus of target cells in these tissues with these antibodies; androgen receptors also were found in the cytoplasm of some target cells. Actinomycin D and 3'-deoxyadenosine, inhibitors of transcription, RNA processing and nucleo-cytoplasmic transport of RNA, interfere with the intracellular dynamics of androgen receptors, suggesting as we have proposed previously that androgen receptors may function not only at the site of transcription but also are involved in posttranscriptional regulation of mRNA stability and utilization.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of human TR3 receptor: a member of steroid receptor superfamily.

Complementary DNAs (cDNAs) encoding a member of steroid receptor super-family, named TR3 receptor, were isolated from a human prostate lambda gt11 cDNA library on the basis of homology of oligonucleotide probes to the DNA-binding domain common to members of the steroid receptor super-family. Expression of TR3 receptor cDNA produced a 64 kDa DNA-binding protein in a rabbit reticulocyte lysate. Nucleotide sequence analysis showed that TR3 receptor cDNA contains two regions of sequences which correspond to the DNA- and hormone-binding domains of members of the steroid receptor super-family. The amino acid sequences in the hormone-binding domain of the TR3 receptor shares about 20% homology with estrogen receptor and less than 15% homology with other known steroid receptors. The DNA-binding domain of the TR3 receptor has about 55% homology with all other known steroid receptors. TR3 receptor had 86% nucleotide and 91% amino acid sequence homology with mouse NUR/77, suggesting that TR3 receptor may be a human homologue of mouse NUR/77 gene product.

Amino Acid Sequence↗
PubMed1989

Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies.

Complementary DNA segments that encode different domains of human and rat androgen receptors were fused to the Escherichia coli trpE gene using pATH expression vectors. Fusion proteins expressed by the bacteria were used to immunize rats and rabbits to obtain polyclonal antibodies to androgen receptors. Spleen cells of immunized rats were fused with myeloma cells to obtain stable hybridomas that produced monoclonal antibodies. Gradient centrifugation and immuno-precipitation assays indicated that the antibodies interacted with androgen receptors specifically.

Amino Acid Sequence↗
PubMed1988

Identification of a new member of the steroid receptor super-family by cloning and sequence analysis.

We have isolated a human testis complementary DNA clone on the basis of homology to the DNA binding domain of steroid receptors. Expression of this complementary DNA, which we call TR2, produces a 52 kd DNA binding protein that does not bind significantly to any known steroids. Northern blot analysis has shown that TR2 mRNA is about 2.5 kilobases (kb) and is relatively abundant in androgen-sensitive organs, such as ventral prostate and seminal vesicle. Dot blot hybridization indicates that TR2 mRNA levels increased after castration of rats, and this increase is reversed by 5 alpha-dihydrotestosterone injection. This evidence suggests that TR2 mRNA levels are negatively controlled by androgen in the rat ventral prostate.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of human and rat complementary DNA encoding androgen receptors.

Complementary DNAs (cDNAs) encoding androgen receptors were obtained from human testis and rat ventral prostate cDNA libraries. The amino acid sequence deduced from the nucleotide sequences of the cDNAs indicated the presence of a cysteine-rich DNA-binding domain that is highly conserved in all steroid receptors. The human cDNA was transcribed and the RNA product was translated in cell-free systems to yield a 76-kilodalton protein. The protein was immunoprecipitable by human autoimmune antibodies to the androgen receptor. The protein bound androgens specifically and with high affinity.

Amino Acid Sequence↗
PubMed1988

Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors.

Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural ARs. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens.

Amino Acid Sequence↗