PTEN-mediated G1 cell-cycle arrest in LNCaP prostate cancer cells is associated with altered expression of cell-cycle regulators.

BACKGROUND: The tumor suppressor PTEN regulates many biological processes. A well-known downstream effector of PTEN is phospho-Akt. Although PTEN is the most frequently inactivated gene in prostate cancer, its mode of action is not fully understood. We studied the association of regulated PTEN expression with changes in biological function and gene expression profiles. METHODS: PTEN-negative LNCaP cells were stably transfected with wild-type PTEN cDNA under inducible control, resulting in LNCaP/PTEN cells. Microarray analysis was used to monitor gene expression changes upon induction of PTEN. Expression of selected individual genes was studied in Q-PCR and siRNA experiments. Cell-cycle distribution was analyzed by flow cytometry. RESULTS: Induced expression of PTEN in LNCaP/PTEN cells significantly inhibited cell proliferation, at least partly due to cell-cycle arrest at the G1 phase. Expression profiling combined with pathway analysis revealed that PTEN-dependent G1 growth arrest was associated with an altered mRNA expression of the G1 cell-cycle regulators Cdc25a, E2F2, cyclin G2, and RBL2/p130. Specific inhibition of Akt signaling by siRNA resulted in downregulation of both E2F2 and Cdc25a mRNA expression and upregulation of the FOXO target cyclin G2, similar to the effect observed by PTEN induction. However, Akt did not mediate the PTEN-dependent RBL2/p130 mRNA expression in LNCaP/PTEN cells. CONCLUSIONS: The results indicate that PTEN dependent gene expression is important in cell-cycle regulation and is mediated by both Akt-dependent and -independent mechanisms.

Both androgen receptor and glucocorticoid receptor are able to induce prostate-specific antigen expression, but differ in their growth-stimulating properties of LNCaP cells.

Androgen receptor-positive LNCaP cells were stably transfected with a rat glucocorticoid receptor (GR) expression plasmid. Ligand-binding studies in the generated cell lines revealed high-affinity binding of the cognate ligands to their receptors. Transfection experiments with the newly derived cell lines showed that, like androgen receptor, GR can induce activity of a prostate-specific antigen promoter fragment linked to the luciferase gene. Similarly, dexamethasone can stimulate expression of endogenous prostate-specific antigen messenger RNA. Cell proliferation could be induced by R1881. In contrast, dexamethasone treatment of the GR-positive sublines had no stimulatory effect on cell growth. Using the differential display technique, a so far unknown complementary DNA fragment, designated 21.1, specifically induced by androgens and not by glucocorticoids, has been identified. In conclusion, the newly generated cell lines, together with the parental LNCaP cell line, form an attractive system with which to study the mechanism of specificity of steroid hormone regulation of gene expression.

Androgen receptor mutations.

Male sexual differentiation and development proceed under direct control of androgens. Androgen action is mediated by the intracellular androgen receptor, which belongs to the superfamily of ligand-dependent transcription factors. At least three pathological situations are associated with abnormal androgen receptor structure and function: androgen insensitivity syndrome (AIS), spinal and bulbar muscular atrophy (SBMA) and prostate cancer. In the X-linked androgen insensitivity syndrome, defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. Complete or gross deletions of the androgen receptor gene have not been found frequently in persons with complete androgen insensitivity syndrome. Point mutations at several different sites in exons 2-8 encoding the DNA- and androgen-binding domain, have been reported for partial and complete forms of androgen insensitivity. A relatively high number of mutations were reported in two different clusters in exon 5 and in exon 7. The number of mutations in exon 1 is extremely low and no mutations have been reported in the hinge region, located between the DNA-binding domain and the ligand-binding domain and which is encoded by the first half of exon 4. Androgen receptor gene mutations in prostate cancer are very rare and are reported only in exons 4-8. The X-linked spinal and bulbar muscle atrophy (SBMA; Kennedy's disease) is associated with an expanded length (> 40 residues) of one of the polyglutamine stretches in the N-terminal domain of the androgen receptor.

The human androgen receptor: structure/function relationship in normal and pathological situations.

Discrete functions have been attributed to precise regions of the human androgen receptor (hAR) by expression of deletion mutants in COS and HeLa cells. A large C-terminal domain constitutes the hormone-binding region and a central basis, cysteine-rich domain is responsible for DNA binding. In addition, separate domains responsible for transactivation and nuclear translocation have been identified. In LNCaP cells (a prostate tumor cell line) the hAR is a heterogeneous protein which is synthesized as a single 110 kDa protein, but becomes rapidly phosphorylated to a 112 kDa protein. Metabolic labeling experiments using radioactive orthophosphate also indicated that the hAR is a phosphoprotein. Structural analysis of the AR gene in LNCaP cells and in 46, XY-individuals displaying androgen insensitivity (AIS) has revealed several different point mutations. In LNCaP cells the mutation affects both binding specificity and transactivation by different steroids. In a person with complete AIS a point mutation was identified in the splice donor site of intron 4, which prevents normal splicing and activates a cryptic splice donor site in exon 4. The consequence is a functionally inactive AR protein due to an in-frame deletion in the steroid-binding domain. In two unrelated individuals with complete AIS, two different single nucleotide alterations in codon 686 (Asp) were found. Both mutations resulted in functionally inactive ARs due to rapidly dissociating hormone-AR complexes. It is concluded that the hAR is a heterogeneous phosphoprotein in which functional errors have a dramatic impact on phenotype and fertility of 46, XY-individuals.

Functional domains of the human androgen receptor.

A series of human androgen receptor (AR) deletion mutants was constructed to study the relationship between various structural domains and their different functions in the AR protein. Immunoblots of wild type AR and AR mutants expressed in COS-1 cells, revealed a doublet appearance of all AR proteins. One exception was an AR mutant lacking amino acid residues 51-211 that migrated as a single protein band, possibly due to altered post-translational modification. The steroid binding domain was found to be encoded by approx. 250 amino acid residues in the C-terminal end. Deletions and truncations in this part of the receptor abolished hormone binding. The N-terminal domain was observed to be essential for transcriptional activation. AR mutants lacking large parts of this domain were transcriptionally inactive. Deletion of the hormone binding domain yielded a constitutively active AR protein, indicating that in the absence of hormone this domain displays an inhibitory function. In the absence of ligand the wild type AR expressed in COS-1 cells was distributed over nucleus and cytoplasm. The addition of hormone directed all androgen receptors to the nucleus. In contrast, an AR mutant lacking part of the DNA binding domain and part of the hinge region, was almost exclusively cytoplasmic in the absence of hormone. This mutant lacks a conserved region, homologous to the SV40 large T- and nucleoplasmin nuclear localization signal. Hormone induced transfer of this AR mutant to the nucleus, indicating the presence of a second, hormone dependent nuclear targeting mechanism.

The promoter of the prostate-specific antigen gene contains a functional androgen responsive element.

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.

The androgen receptor: functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines.

The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT codon encoding a threonine residue to GCT, encoding alanine.

Variation of prostate-specific antigen expression in different tumour growth patterns present in prostatectomy specimens.

A series of 55 randomly chosen radical prostatectomy specimens was analyzed for expression of prostate-specific antigen (PSA) by immunohistochemical techniques. Tissue sections were selected in such a manner that in addition to glandular benign prostatic hyperplasia (BPH), one or more different prostatic tumour growth patterns were present. Four monoclonal antibodies, directed against three different PSA epitopes, and one polyclonal anti-PSA antiserum were used. Expression of PSA was compared with that of prostate-specific acid phosphatase (PAP), recognized by two different polyclonal antisera. A critical dilution aimed at a maximum of staining intensity on BPH tissue sections was chosen for all antibodies. Anti-PSA and anti-PAP antisera stained essentially all BPH samples (over 90%). Irrespective of the nature of the antibodies used, PSA expression was found to be decreased in prostatic carcinoma. A clear cut relationship was found between immunoreactivity for PSA and the degree of differentiation of the tumour area. Under the experimental conditions used the PSA monoclonal antibodies stained only 1 out of 10 undifferentiated carcinomas, whereas 50% to 70% of the well- and moderately-differentiated carcinomas showed immunoreactivity. This correlation was less pronounced with the PAP staining pattern. If the PSA antibody titer was raised the percentage of clearly staining undifferentiated carcinomas could be considerably increased (up to 60%-100%), indicating that PSA expression is not absent, but lowered in most (if not all) undifferentiated carcinomas.

Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor.

Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.

Structural organization of the human androgen receptor gene.

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.

The prostate-specific antigen gene and the human glandular kallikrein-1 gene are tandemly located on chromosome 19.

Using a prostate-specific antigen cDNA as a hybridization probe, clones containing the kallikrein genes encoding prostate-specific antigen, human glandular kallikrein-1 and pancreas/kidney kallikrein were isolated from a human genomic library. Clones containing the prostate-specific antigen gene and the human glandular kallikrein-1 gene overlap and span a region of about 36 kb. The two genes are aligned in a head to tail orientation at a mutual distance of 12 kb. Southern blot analysis of DNA from a panel of human-hamster hybrid cells with specific probes revealed the genes to be situated on chromosome 19. Assuming that the pancreas/kidney kallikrein gene is located in the same cluster, the distance to the prostate-specific antigen gene and the human glandular kallikrein gene must be at least 15 kb.

Characterization of the prostate-specific antigen gene: a novel human kallikrein-like gene.

Using Prostate-specific Antigen cDNA fragments as hybridization probes a clone containing the information for the gene encoding Prostate-specific Antigen was isolated form a human genomic DNA library. The complete gene (about 6 kb) was sequenced and shown to be composed of four introns and five exons. Two major transcription initiation sites were found. The sequence of the promoter region revealed the presence of various well known transcription regulatory elements including a TATA box. A high percentage of homology was found between the Prostate-specific Antigen gene and the hGK-1 gene (82%). This homology extended into the promoter region. Two previously described variant Prostate-specific Antigen cDNAs can now be explained by intron retention and alternative splicing of the primary transcript.

The N-terminal domain of the human androgen receptor is encoded by one, large exon.

Using specific cDNA hybridization probes, the first coding exon of the human androgen receptor gene was isolated from a genomic library. The exon contained an open reading frame of 1586 bp, encoding an androgen receptor amino-terminal region of 529 amino acids. The deduced amino acid sequence was characterized by the presence of several poly-amino acid stretches of which the long poly-glycine stretch (16 residues) and the poly-glutamine stretch (20 residues) were most prominent. Androgen receptor cDNAs from different sources contained information for poly-glycine stretches of variable size (23 and 27 residues, respectively). The androgen receptor amino-terminal domain was found to be hydrophilic and have a net negative charge. Combined with the previously described, partially overlapping cDNA clone 7A2M27 (Trapman et al. (1988) Biochem. Biophys. Res. Commun. 153, 241-248), the complete human androgen receptor was deduced to have a size of 910 amino acids.

Structure and function of the androgen receptor.

The androgen receptor in several species (human, rat, calf) is a monomeric protein with a molecular mass of 100-110 kDa. The steroid binding domain is confined to a region of 30 kDa, while the DNA-binding domain has the size of approx. 10 kDa. A 40 kDa fragment containing both the DNA and steroid binding domain displayed a higher DNA binding activity than did the intact 100 kDa molecule. cDNA encoding the major part of the human androgen receptor was isolated. The cDNA contains an open reading frame of 2,277 bp but still lacks part of the 5'-coding sequence. Homology with the progesterone and glucocorticoid receptor was about 80% in the DNA binding domain and 50% in the steroid binding domain. The present data provide evidence that the androgen receptor belongs to the superfamily of ligand responsive transcriptional regulators and consists of three distinct domains each with a specialized function.

The human androgen receptor: domain structure, genomic organization and regulation of expression.

The domain structure and the genomic organization of the human androgen receptor (hAR) has been studied after molecular cloning and characterization of cDNA and genomic DNA encoding the hAR. The cDNA sequence reveals an open reading frame of 2751 nucleotides encoding a protein of 917 amino acids with a calculated molecular mass of 98,845 D. The N-terminal region of the hAR is characterized by a high content of acidic amino acid residues and by several homopolymeric amino acid stretches. The DNA-binding domain showed a high homology with the DNA-binding domain of the human glucocorticoid receptor (hGR) and the human progesterone receptor (hPR). The predominantly hydrophobic steroid binding domain of the hAR is 50-55% homologous with the ligand binding domains of the hGR and hPR. Transient expression of recombinant AR cDNA in COS-cells resulted in the production of a 110 kDa protein with the expected binding specificity of androgen receptors. Co-transfection with a reporter-gene construct [CAT(chloramphenicol acetyl transferase) under direction of the androgen regulated MMTV-promoter] showed that the protein is functionally active with respect to transcription regulation. In the LNCaP prostate carcinoma cell line two major (11 and 8 kb) and one minor (4.7 kb) mRNA species can be found which can be down-regulated by androgens. The hAR protein coding region was shown to be divided over eight exons with an organization similar to that of the progesterone and oestrogen receptor. The sequence encoding the N-terminal domain was found in one large exon. The two DNA-binding fingers were encoded by two small exons; the information for the androgen-binding domain was found to be distributed over five exons. Southern blot analysis of genomic DNA revealed that the hAR is encoded by one single gene, which is situated on the X-chromosome.

Molecular cloning and characterization of novel prostate antigen cDNA's.

Three different prostate antigen cDNA's were isolated from a PC 82 prostate tumor cDNA library. PA 75 has a size of 1.4 kb and contains the almost complete information for the 35 kD prostate antigen preproprotein. The 1.6 kb PA 525 cDNA lacks about 0.2 kb of the 3'-non coding region and contains an additional internal 0.4 kb fragment as a result of alternative splicing. PA 424 represents a 0.6 kb variant of PA 75. It contains a 0.15 kb internal fragment and a poly(A) tail preceded by an AAGAAA motive at the 3'-end. The predicted protein products of PA 525 and PA 424 will be different from PA 75 at the C-terminal end. In RNA preparations of two human prostate tumors (PC 82 and PC EW) seven different prostate antigen transcripts can be detected ranging in size from 0.5 kb to 5.6 kb. PA 75 cDNA represents the major 1.5 kb mRNA. PA 424 correlates with a 0.9 kb transcript and PA 525 with a 1.9 kb mRNA species.

Cloning, structure and expression of a cDNA encoding the human androgen receptor.

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.

Expression of cellular oncogenes in human prostatic carcinoma cell lines.

Prostatic cancer is one of the most frequent forms of malignancy in Western countries. Initially, growth of the majority of prostate tumors can be manipulated by endocrine therapy. However, ultimately androgen independent tumors continue to grow. We studied the expression of oncogenes in four different human prostatic carcinoma cell lines: PC 3, PC 133, PC 135, which are androgen independent, and the hormone dependent PC 82 cell line. Large amounts of Ha-ras and myc mRNA were present in all cell lines. Transcripts of fes, int-1 and abl were never detected. In some of the cell lines the presence of N-ras, Ki-ras, myb, fos, fms and sis mRNA was observed. The PC 82 cell line showed, in addition to myc and Ha-ras high levels of fos expression. Inhibition of tumor cell proliferation by withdrawal of androgen was accompanied by a tenfold reduction of the fos mRNA level and a twofold reduction of Ha-ras transcripts. In contrast, the expression of myc was not changed.

Mouse interferon alpha and beta genes are linked at the centromere proximal region of chromosome 4.

In order to determine the chromosomal localization of the murine interferon-alpha (MuIFN-alpha) and murine interferon-beta (MuIFN-beta) genes the DNAs of a panel of somatic cell hybrids were analysed by Southern blot hybridization. The hybrid cells were derived from E36 Chinese hamster cells and GRSL or GR MaTu mouse cells and retained all hamster chromosomes but segregated mouse chromosomes. The MuIFN-alpha probe used was a 0.7 kb HindIII-EcoRI fragment derived from the MuIFN-alpha 1 gene which hybridized with both mouse and hamster DNA. However, four fragments present in EcoRI digests of mouse DNA were clearly absent from the hybridization profile of EcoRI-digested hamster DNA and could be used for detection of MuIFN-alpha sequences in the hybrid cells. The MuIFN-beta probe, a 0.5 kb BglII-BamHI fragment derived from the MuIFN-beta gene, hybridized with a 2.6 kb EcoRI fragment of mouse DNA and only weakly cross-hybridized with a 4.8 kb EcoRI fragment in hamster DNA. Southern blot analysis of DNA from mouse/hamster hybrids compared with the analysis of chromosome markers showed that both the MuIFN-alpha and the MuIFN-beta genes are located on chromosome 4. Analysis of DNA from hybrids that contained only part of chromosome 4 indicated that the MuIFN-alpha gene family and the MuIFN-beta gene are situated at the centromere-proximal region of the chromosome.

Isolation and characterization of subspecies of murine interferon alpha.

Interferon produced by mouse L-929 cells by incubation with poly(rI).poly(rC) is known to be composed of a mixture of MuIFN-alpha and MuIFN-beta. The alpha component was separated from the bete species by affinity chromatography over a monoclonal anti-MuIFN-beta agarose column and partially purified by gel filtration. MuIFN-alpha, prepared by this method was separated into at least five subspecies by chromatofocusing. The approximate pI values of these components are greater than or equal to 7.5, 6.5, 6.2, 5.9 and 5.6, respectively. Component 3 (pI 6.2) was the most prominent subspecies present in our MuIFN-alpha preparations, representing 40 to 50% of the total antiviral activity. Component 1 (pI greater than or equal to 7.5) which accounted for about 5% of the antiviral activity on mouse cells, differed in some properties from the other interferon subspecies. It showed a relatively high antiviral activity on heterologous cells and it was eluted from a Sephadex column after the other alpha subspecies. Furthermore, it showed a diminished binding to heparin as compared to the other MuIFN-alpha subspecies, indicating a lower affinity for polynucleotides.