Frequent inactivation of PTEN in prostate cancer cell lines and xenografts.

Loss of chromosome 10q is a frequently observed genetic defect in prostate cancer. Recently, the PTEN/MMAC1 tumor suppressor gene was identified and mapped to chromosome 10q23.3. We studied PTEN structure and expression in 4 in vitro cell lines and 11 in vivo xenografts derived from six primary and nine metastatic human prostate cancers. DNA samples were allelotyped for eight polymorphic markers within and surrounding the PTEN gene. Additionally, the nine PTEN exons were tested for deletions. In five samples (PC3, PC133, PCEW, PC295, and PC324), homozygous deletions of the PTEN gene or parts of the gene were detected. PC295 contained a small homozygous deletion encompassing PTEN exon 5. In two DNAs (PC82 and PC346), nonsense mutations were found, and in two (LNCaP and PC374), frame-shift mutations were found. Missense mutations were not detected. PTEN mRNA expression was clearly observed in all cell lines and xenografts without large homozygous deletions, showing that PTEN down-regulation is not an important mechanism of PTEN inactivation. The high frequency (60%) of PTEN mutations and deletions indicates a significant role of this tumor suppressor gene in the pathogenesis of prostate cancer.

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.

Isolation, properties and chromosomal localization of four closely linked hamster interferon-alpha-encoding genes.

Three recombinant phages containing hamster interferon-alpha-encoding genes (Ha Ifa) were isolated from a Ha genomic library, using a murine (Mu) Ifa probe. The phage inserts contained overlapping genomic fragments which span a total length of approx. 30 kb, on which four Ha Ifa genes are localized. The Ifa gene cluster could be assigned to hamster chromosome 2q. The nt sequences of the four Ifa genes were determined. Two of the genes are functional (Ha Ifa-1 and Ifa-3) and two are pseudogenes (Ifa-ps2 and Ifa-ps4). Ha Ifa-1 and -3 were transiently expressed in COS cells and they gave rise to protein products (A1 and A3, respectively) with antiviral properties on hamster CHO cells. In addition, Ha A1 revealed high antiviral activity on murine L929 cells.

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.

Identification and androgen-regulated expression of two major human glandular kallikrein-1 (hGK-1) mRNA species.

The screening of an oligo(dT)-primed prostate cDNA library with a human glandular kallikrein-1 (hGK-1) genomic DNA fragment resulted in the isolation of two different hGK-1 cDNAs. A 1.2 kb cDNA (pGK-1) contains an open reading frame of 510 bp, encoding the major part of the previously predicted hGK-1 protein (Schedlich et al. (1987) DNA 6, 429-437). This cDNA contains a 3'-untranslated region of 677 nucleotides and terminates in a poly(A) stretch, preceded by the canonical AATAAA polyadenylation signal. A second cDNA (pGK-10A), with a size of 1.5 kb, contains an open reading frame of 669 nucleotides preceded by 16 nucleotides of the 5'-untranslated region. pGK-10A differs from pGK-1 by the presence of an additional 37 bp fragment, interrupting the protein coding region of hGK-1, which results from the use of an alternative splice donor site of intron IV of the hGK-1 gene. The mature protein (excluding presumed pre- and propeptides) as deduced from the pGK-10A cDNA sequence, has a size of 199 amino acids and differs at the COOH-terminus from the 237 amino acid hGK-1 protein. The alternatively spliced mRNA comprises approximately 20% of the hGK-1 transcripts, as deduced from analysis of mRNA from prostate cells by PCR amplification of specific fragments. The regulation of hGK-1 mRNA expression was studied in different human prostate tumors and cell lines by Northern blotting, using a hGK-1-specific oligonucleotide probe. A high level of hGK-1 expression was found in the androgen-dependent tumors PC 82 and PC EW. hGK-1 mRNA was also present in the androgen-sensitive LNCaP cell line, but undetectable in the androgen-insensitive prostate tumors PC 133, PC 135 and the PC 3 cell line. In LNCaP cells, the expression of hGK-1 mRNA was strongly induced by androgens. Regulation of expression of the closely related prostate-specific antigen (PA) gene showed a similar pattern.

Characterization of the human kallikrein locus.

The human kallikrein gene family is composed of three members: tissue kallikrein (KLK1), prostate-specific antigen (PA or APS), and human glandular kallikrein-1 (hGK-1 or KLK2). The three genes have previously been isolated and mapped to chromosome 19q13.2-q13.4. Further analysis of an area of 110 kb surrounding the kallikrein genes by CHEF electrophoresis and chromosome walking showed clustering of the three genes. The KLK1 gene is positioned in the opposite orientation of the APS and KLK2 genes in the order KLK1-APS-KLK2. The APS and KLK2 gene are separated by 12 kb; the distance between KLK1 and APS is 31 kb. A CpG island was detected in the region between KLK1 and APS. Preliminary data indicate that this CpG island is located directly adjacent to a gene that is unrelated to the kallikreins and seems to be ubiquitously expressed.

The interferon-stimulated gene 54 K promoter contains two adjacent functional interferon-stimulated response elements of different strength, which act synergistically for maximal interferon-alpha inducibility.

The interferon-alpha(IFN-alpha)-regulated hamster ISG-54 K gene, which is activated in hamster CHO-12 cells at least 40-fold, was isolated and the promoter region was characterized in detail. Sequence analysis revealed the presence of two elements, closely related to the interferon-stimulated-response-element (ISRE) consensus sequence [AGTTTCNNTTTC(CT)]. The putative ISRE-I sequence (GGTTTCAATTTCT) is located at position -97 to -85; ISRE-II (AGTTTTACTTTCT), which differs at three positions from ISRE-I, is found directly upstream of ISRE-I at position -110 to -98. In a transient transfection assay in CHO-12 cells the wild-type hamster ISG-54K-promoter-chloramphenicol-acetyl-transferase (CAT) reporter construct showed a 40-80-fold induction, offering an excellent model to study the functional properties of the two ISRE. To find out whether both elements were functional in interferon regulation of the promoter, selected point mutations were introduced in the -110 to -85 region and in flanking sequences. The (mutated) ISG-54 K promoter was linked to the CAT reporter gene and transiently expressed in CHO cells in the absence and presence of murine (Mu)IFN-alpha 6. Transfections showed that both the -97 to -85 (ISRE-I) and the -110 to -98 (ISRE-II) segment were needed for optimal interferon induction of the ISG-54 K promoter. However, ISRE-I has an approximately sevenfold stronger activity compared to ISRE-II. Sequential substitution of the three ISRE-I bases, which differ in ISRE-II showed that the T at position -105 causes the lower activity of ISRE-II. Transfection of ISG-54 K promoter constructs, in which ISRE-I was replaced by ISRE-II, which generates a promoter with two ISRE-II segments, and vice versa (two ISRE-I), provided further evidence for a role of both elements in IFN-alpha induction. Importantly, all data obtained in transfection studies show that the two ISRE cooperate synergistically. The mechanism of synergism is most probably an indirect interaction between transcription factors binding to the ISRE, because an increase in the spacial arrangement of the two ISRE with a complete helical turn or half a turn did not result in a substantial decrease in promoter activity.

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.

Structure, chromosome localization, and regulation of expression of the interferon-regulated mouse Ifi54/Ifi56 gene family.

The interferon-alpha (IFN-alpha) regulated mouse Ifi54/Ifi56 gene family, which is composed of at least four members (Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2), was isolated and characterized. In addition, the chromosomal localization of the four genes was determined. The Ifi54 and Ifi56 genes show an identical organization. Both are composed of a very small first exon and a second exon, which contains the complete open reading frame, except for the ATG start codon and the first two nucleotides of the second codon. In both genes, the two exons are separated by a small intron (5 and 2.5 kb, respectively). Expression of both genes is rapidly induced by IFN-alpha (within 2 h). The Ifi54 promoter region contains two sequences, which are closely related to the interferon stimulated response element (ISRE) consensus sequence (ISRE 1, GGTTTCAATTTCT, and ISRE 2, AGTGTTACTTTCT). The two elements are located directly adjacent to each other. A similar organization was recently established for the hamster Ifi54 promoter (Bluyssen et al., 1994). However, the mouse promoter is 70% less active than the hamster promoter. It turned out that ISRE 2 is hardly active, due to the G at position 4, which is a T in the hamster Ifi54 ISRE 2 and in the ISRE consensus sequence. The Ifi56 promoter region contains at a similar position two functional ISREs of identical strength (ISRE 1, AGTTTCAGTTTCT, and ISRE2, AGTTTCACTTTCC). In the Ifi56 promoter, the two ISRE motifs are separated by 6 bp. In addition to the Ifi56 gene, parts of two closely related genes (Ifi56-ps1 and Ifi56-ps2) were isolated. Both fragments contain an Ifi56-related open reading frame. However, we were unable to isolate the presumed first exon of Ifi56-ps1 and Ifi56-ps2, nor could we show expression of the genes. The Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2 genes could all be assigned to mouse chromosome 19D1, suggesting a tight clustering.