High-throughput genotyping of high-risk HPV by the digene HPV Genotyping LQ Test using GP5+/6+-PCR and xMAP technology.

BACKGROUND: Epidemiologic studies have classified 18 genotypes of the human papillomavirus (HPV) as (probably) high-risk (HR) based on their association with cervical cancer, i.e., HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. Given the fact that certain HR HPV types confer an increased risk of cervical (pre)cancer, type-specific identification might aid clinical management of women tested positive for HR HPV. Therefore, the development of robust, high-throughput genotyping assays is important. OBJECTIVES: An analytical comparison of the digene HPV Genotyping LQ Test (digene LQ Test), capable of identifying 18 HR types using bead-based xMAP suspension array technology, with the established Reverse Line Blot (RLB) genotyping assay was carried out on amplimers generated with the clinically validated GP5+/6+-PCR method. STUDY DESIGN: GP5+/6+ amplimers, generated from 434 digene High Risk HPV HC2 DNA Test (HC2)-positive and 95 HC2-negative cervical smears, were genotyped by both the digene LQ Test and the RLB genotyping assay. RESULTS: The genotyping assays revealed high agreement for overall HR HPV detection (ú = 0.884) and type-specific identification of the 18 HR HPV types (overall ú = 0.958, individual ú range 0.795 to 1.000). The digene LQ Test demonstrated a very good inter-laboratory reproducibility (ú = 0.987). Among the HC2-positive women, the digene LQ Test revealed positivity for one or more HR HPV type(s) in 85.9%, and negativity was observed in 97.9% of the HC2-negative women. CONCLUSIONS: The digene LQ Test demonstrated a high genotyping agreement with the established RLB genotyping assay on GP5+/6+ amplimers. This novel assay allows for high-throughput genotyping following HR HPV testing by HC2.

A novel gene on human chromosome 2p24 is differentially expressed between androgen-dependent and androgen-independent prostate cancer cells.

Identification of genes involved in the transition from androgen-dependent to androgen-independent prostate cancer is important to extend our current knowledge of the disease. Using differential display RT-PCR analysis between androgen-dependent and androgen-independent prostate cancer cells, we have identified a novel gene, designated GC109. GC109 harbours a putative Cys-His cluster, a nuclear localisation signal, a leucine zipper and a ret finger protein (rfp)-like domain. GC109 mRNA expression in normal human tissues was found not to be restricted to the prostate. However, using a variety of 15 human cancer cell lines, GC109 mRNA was preferentially expressed in androgen-dependent LNCaP-FGC, compared with androgen-independent LNCaP-LNO, DU145 and PC3 human prostate cancer cells. Finally, the GC109 gene was mapped on human chromosome 2p24. Based on its protein domain structure and chromosomal localisation, we hypothesise that GC109 may be involved in chromosomal rearrangements in prostate cancer.

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells.

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.

Identification of a gene on human chromosome 8q11 that is differentially expressed during prostate-cancer progression.

Using differential-display RT-PCR analysis between androgen-dependent LNCaP-FGC and androgen-independent LNCaP-LNO human prostate-cancer cells, we have identified a gene not previously described as being expressed in prostate. The gene is more highly expressed in androgen-independent than in androgen-dependent LNCaP prostate-cancer cells. Sequence analysis showed that the gene has already been cloned as a transcript present in embryonic brain, with unknown functions. Expression of the gene was found not to be restricted to the prostate, and not regulated by androgens in androgen-independent prostate-cancer cells. In concert with the cell-culture system, Northern-blot analysis of gene expression in vivo, using a panel of human prostate-cancer xenografts, demonstrated that the gene is more highly expressed in androgen-independent than in androgen-dependent prostate-cancer xenografts. The gene could be mapped on human chromosome 8q11. The 8q arm is known to be frequently amplified during prostate-cancer progression and harbors several proto-oncogenes potentially involved in cancer development. Since expression of the gene is positively correlated with prostate-cancer progression and its 8q11 chromosomal localization, we hypothesize that the gene may be involved in the development and progression of prostate cancer.

Identification of a homozygous deletion at 8p12-21 in a human prostate cancer xenograft.

One of the most frequent genetic abnormalities in prostate cancer is loss of the complete or part of the short arm of chromosome 8, indicating the localization of one or more tumor suppressor genes on this chromosomal arm. Using allelotyping, a frequently deleted region in prostate cancer in a genetic interval of approximately 17 cM between sequence tagged sites D8S87 and D8S133 at chromosome arm 8p12-21 was previously detected. A detailed physical map of this region is now available. Using known and novel polymorphic and nonpolymorphic sequence tagged sites in this interval, a search for homozygous deletions in DNAs from 14 prostate cancer-derived cell lines and xenografts was carried out. In DNA from xenograft PC133, the presence of a small homozygously deleted region of 730-1,320 kb was unambiguously established. At one site, the deletion disrupts the Werner syndrome gene. Data from allelotyping were confirmed and extended by fluorescence in situ hybridization analysis of PC133 chromosome spreads using centromere, YAC, and PAC chromosome 8 probes.

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.

Chromosome region 8p11-p21: refined mapping and molecular alterations in breast cancer.

Several genes, most of them unknown, of the short arm of chromosome 8 are involved in malignant diseases. Numerous studies have implicated a portion of the 8p11-p21 region as the location of one or more tumor suppressor genes involved in a variety of human cancers, including breast cancer. We and others have reported linkage analyses suggesting the presence of a putative breast cancer susceptibility gene. Furthermore, several oncogenes of the 8p11-p12 region are involved in reciprocal translocations in myeloproliferative and myelodysplastic disorders and in amplification in breast cancer. To facilitate the analysis of the 8p11-p21 region and the cloning of candidate oncogenes and tumor suppressor genes, a high-resolution physical and transcriptional map was established with 39 yeast artificial chromosomes and 94 markers, including so-called sequence-tagged sites and expressed sequence-tagged sites derived from either known genes or expressed sequence tags corresponding to unidentified transcripts. In addition, four novel transcripts were identified and localized precisely within the map. This transcription map provides a detailed description of gene order for the 8p11-p21 region and will be helpful in the identification of candidate genes for diseases. From this basis, we refined the mapping of two types of molecular alterations that occur at 8p11-p21 in sporadic breast cancers, i.e., amplification and deletion.

Mouse ubiquitous mitochondrial creatine kinase: gene organization and consequences from inactivation in mouse embryonic stem cells.

Individual members of the creatine kinase isoenzyme family (CK; EC 2.7.3.2), which play a prominent role in energy homeostasis, are encoded by four separate nuclear genes. We have isolated and characterized the complete mouse UbCKmit gene, the product of which is ubiquitously expressed and is located in the intermembrane space of mitochondria. Transcription of this gene is initiated at multiple adjacent positions and the region immediately upstream of these sites shares many features with genes encoding housekeeping proteins. These include a high G/C content, absence of TATA and CCAAT motifs, and presence of SP1 and AP2 recognition sequences. In addition, a binding site for HIP1, hormone-responsive elements, and three Mt-motifs, known as boxes shared between nuclear genes encoding mitochondrial proteins, were identified. To study the functional role of the UbCKmit protein, we have inactivated both UbCKmit alleles in mouse embryonic stem (ES) cells. UbCKmit-deficient cells, obtained by consecutive rounds of gene targeting using homologous recombination and drug selection-driven gene conversion events, show no obvious growth disadvantage or abnormal differentiation potential. Activities of mitochondrial cytochrome c oxidase and citrate synthase, as well as the rate of pyruvate oxidation, showed values equal to wild-type cells, indicating a normal aerobic metabolism. Mitochondria of in vivo differentiated knock-out cells were structurally intact, as demonstrated by electron microscopy. Approaches to study the role of the UbCKmit gene further are discussed.

Lysophosphatidic acid and bradykinin have opposite effects on phenotypic transformation of normal rat kidney cells.

The bioactive lipid lysophosphatidic acid is besides a strong mitogen for quiescent fibroblasts, a potent inducer of phenotypic transformation of normal rat kidney cells. The lysophosphatidic acid induced loss of density-arrest is strongly inhibited by bradykinin. Although their effects on normal rat kidney cell proliferation are opposite, bradykinin mimics many of the intracellular effects induced upon lysophosphatidic acid receptor activation, including phosphoinositide turnover, Ca(2+)-mobilization and arachidonic acid release. Bradykinin does not counteract the lysophosphatidic acid induced reduction of cAMP levels in normal rat kidney cells. However, bradykinin inhibits the lysophosphatidic acid and other growth factor induced phenotypic transformation through the induction of a so far uncharacterized prostaglandin G/H synthase product. The growth inhibitory effect of bradykinin is limited to density-arrested cells, while upon prolonged treatment bradykinin itself is capable to induce the loss of density-dependent growth control. It is concluded that bradykinin is a bifunctional regulator of normal rat kidney cell proliferation and that its inhibitory effects are mediated via the induction of a prostaglandin derivative.

Effect of bradykinin on loss of density-dependent growth inhibition of normal rat kidney cells.

Normal rat kidney fibroblasts, density-arrested in the presence of epidermal growth factor (EGF), can be restimulated to proliferate in a synchronous way and acquire a transformed phenotype following treatment with additional growth factors like retinoic acid (RA) and transforming growth factor (TGF)-beta. It was found that bradykinin has a strong inhibitory effect on growth stimulation induced by these factors, an effect which cannot be mimicked by PGF2 alpha. The growth-inhibiting effect can be blocked by inhibitors of cyclo-oxygenase activity, indicating that the relevant second messenger is most likely a prostaglandin. Externally added PGJ2, at a concentration of 10 microM, can mimic the inhibitory effect of bradykinin on the loss of density-arrest induced by RA suggesting that PGJ2 is a possible candidate for being the bradykinin induced growth-inhibiting prostaglandin.