Circulating biomarker tissue kallikrein-related peptidase KLK5 impacts ovarian cancer patients' survival.

BACKGROUND: Effective cancer biomarkers for early detection, prognosis, or therapy response prediction are urgently needed in ovarian cancer. Kallikrein-related peptidases, including KLK5, have been reported to play an important role in the course of the disease. PATIENTS AND METHODS: KLK5 antigen content was determined by enzyme-linked immunosorbent assay in ovarian cancer patients' [FIGO (International Federation of Gynecology and Obstetrics) stages I-IV, n = 52] serum as well as ascitic fluid and compared with KLK5 content in serum of patients with benign ovarian tumors (n = 45). RESULTS: KLK5 antigen content was significantly elevated in the serum of ovarian cancer patients compared with the serum of patients with benign ovarian tumors. Forty-two of 52 ovarian cancer serum samples, 42 of 43 benign ovarian tumor serum samples, and all 41 ascitic fluid samples were KLK5 positive. Elevated KLK5 antigen in serum and ascitic fluid of ovarian cancer patients was a prognostic factor for progression-free survival. CONCLUSIONS: Our data support the finding that ovarian cancer patients release significant amounts of KLK5 into serum and ascitic fluid but KLK5 antigen is low in serum of patients with benign ovarian tumors. Increased serum and ascitic fluid KLK5 levels are associated with poor patient outcome, thus underlining the importance of KLK5 as a biomarker for early detection as well as for disease management in ovarian cancer.

Co-detection of members of the urokinase plasminogen activator system in tumour tissue and serum correlates with a poor prognosis for soft-tissue sarcoma patients.

BACKGROUND: The urokinase plasminogen activator (uPA) system is one of the best-investigated protease systems, both under physiological and pathological conditions, including various types of cancer. However, effects of co-expression of members of the uPA system in soft-tissue sarcoma (STS) patients at the protein level in both tumour tissue and serum have not been investigated yet. METHODS: We examined 82 STS patients for protein levels of uPA, PAI-1and uPAR in tumour tissue and serum by ELISA. RESULTS: A significant correlation between high antigen levels of uPA, PAI-1 or uPAR in tumour tissue, and of uPAR in serum, with poor outcome of STS patients was found for the first time. Most strikingly, we observed an additive effect of combined uPA, PAI-1 or uPAR levels in tumour tissue extracts with uPAR levels in serum on patients' prognosis. High uPA/uPAR, PAI-1/uPAR and uPAR/uPAR antigen levels in tumour tissue/serum were associated with a 5.9-fold, 5.8-fold and 6.2-fold increased risk of tumour-related death (P=0.003, 0.001 and 0.002, respectively) compared with those patients who displayed low levels of the respective marker combination. CONCLUSION: As expression of members of the uPA system in tumour tissue and serum is additively correlated with prognosis of STS patients, our results suggest that combinations of these biomarkers can identify STS patients with a higher risk of tumour-related death.

Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.

We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Müller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin. Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

Protein kinase Cdelta expression in breast cancer as measured by real-time PCR, western blotting and ELISA.

The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCdelta. Currently, it is unclear whether PKCdelta is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCdelta in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cdelta expression at the mRNA level was measured using real-time PCR (n=208) and at protein level by both immunoblotting (n=94) and ELISA (n=98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCdelta but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCdelta concentrations determined by ELISA (for the 78 kDa form, r=0.444, P<0.005, n=91 and for the 160 kDa form, r=0.237, P=0.023, n=91) and with PKCdelta mRNA levels (for the 78 kDa form, r=0.351, P=0.001, n=94 and for the 160 kDa form, r=0.216, P=0.037, n=94). Protein kinase Cdelta mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P=0.007, Mann-Whitney U-test). Increasing concentrations of PKCdelta mRNA were associated with reduced overall patient survival (P=0.004). Our results are consistent with a role for PKCdelta in breast cancer progression.

The intron-containing gene for yeast profilin (PFY) encodes a vital function.

The gene coding for profilin (PFY), an actin-binding protein, occurs as a single copy in the haploid genome of Saccharomyces cerevisiae and is required for spore germination and cell viability. Displacement of one gene copy in a diploid cell by a nonfunctional allele is recessively lethal: tetrad analysis yields only two viable spores per ascus. The PFY gene maps on chromosome XV and is linked to the ADE2 marker. The primary transcript of about 1,000 bases contains an intron of 209 bases and is spliced into a messenger of about 750 bases. The intron was identified by comparison with a cDNA clone, which also revealed the 3' end of the transcript. The 5' end of the mRNA was mapped by primer elongation. The gene is transcribed constitutively and has a coding capacity for a protein of 126 amino acids. The deduced molecular weight of

Hydroxamate-type matrix metalloproteinase inhibitor batimastat promotes liver metastasis.

Overexpression of matrix metalloproteinases (MMPs) facilitates tumor cell invasion. Synthetic MMP inhibitors such as batimastat have been designed to treat cancer. We report that because of batimastat treatment, human breast carcinoma cells metastasized to the liver in nude mice and that an increase of liver metastases of murine T-cell lymphoma cells was observed in syngeneic mice. Batimastat treatment also caused liver-specific overexpression of MMPs-2, -9, and mRNA up-regulation of angiogenesis factors and caspase-1, even in tumor-free animals. Induction of organ-specific side effects need to be taken into account regarding further development and clinical use of synthetic MMP inhibitors.

A yeast gene (BLH1) encodes a polypeptide with high homology to vertebrate bleomycin hydrolase, a family member of thiol proteinases.

We have purified bleomycin hydrolase from yeast (molecular mass 55,000 Da). Using protein sequence-derived degenerate oligonucleotide primers and amplification by polymerase chain reaction, the yeast gene BLH1 was isolated and characterized. The deduced amino acid sequence (483 amino acids) exhibits surprisingly high homology to vertebrate bleomycin hydrolase (43% identical residues and 22% conserved exchanges). It contains three blocks of sequences found conserved in other members of the thiol proteinase family and thought to be associated with the catalytic centre. BLH1 is non-essential under all growth conditions tested. However, in the presence of 3.5 mg bleomycin/ml medium wild-type cells have a slight growth advantage compared to blh1 mutant cells.

The gene LEO1 on yeast chromosome XV encodes a non-essential, extremely hydrophilic protein.

A 5.6 kbp segment of DNA from Saccharomyces cerevisiae chromosome XV has been isolated and sequenced. Genetic and nucleotide sequence analyses revealed that this region is closely linked to the ADE2 marker on chromosome XV and densely packed with genetic information. We show the gene organization of the entire region and report the nucleotide sequence of the gene, LEO1, which occurs in single copy in the haploid genome. The deduced amino acid sequence specifies an extremely hydrophilic protein with pronounced domain structure (molecular mass 53.9 kDa). The gene is constitutively expressed at a low level and is non-essential, as indicated by the absence of a phenotype from gene disruption mutants.

Cytoplasmic and mitochondrial forms of yeast adenylate kinase 2 are N-acetylated.

Yeast major adenylate kinase (Aky2p), encoded by a single gene, occurs in two subcellular compartments, mitochondria and cytoplasm. Only 6-8% of the protein which has no cleavable presequence is imported into the organelle (Bandlow et al. (1988) Eur. J. Biochem. 178, 451-457). In the wild type two AKY2-derived signals (a major and a minor one) were detected by a monospecific antibody after two-dimensional gel electrophoresis and Western blotting. The signals reflected identical electrophoretic mobilities and were absent from an AKY2-disrupted strain suggesting that they were due to differently modified forms of Aky2p. Two similar signals were found in a mutant defective in protein N-acetylation, however, the pI values of both spots were shifted towards alkaline pH by one charge. This indicated that both forms of Aky2p were N-acetylated in the wild type and that their charge difference was not caused by incomplete N-acetylation. This observation furthermore suggested that, in the wild type, two different modifications exist one of which is N-acetylation. The second modification remains unidentified. We analysed the influence of protein N-acetylation on mitochondrial import. Both versions of Aky2p occurred in the cytoplasm and in mitochondria. Their proportion was unchanged in the N-acetylation mutant showing that neither modification affected the efficiency of import of adenylate kinase into mitochondria. It is discussed that N-acetylation occurs during or immediately after translation in the cytoplasm so that import of adenylate kinase may ensue co-translationally.

[Tumor-associated prognostic factors of the plasminogen activator family: determination and clinical value of u-PA, t-PA, PAI-1, and PAI-2]. / Tumorassoziierte Prognosefaktoren der Plasminogenaktivator-Familie. Bestimmung und klinische Wertigkeit von u-PA, t-PA, PAI-1 und PAI-2.

Proteolytic factors belonging t the plasminogen activator family (plasmin, u-PA, t-PA, u-PAR, PAI-1, and PAI-2), which usually are involved in blood clotting and degradation of blood clots, are also present in healthy and diseased tissue of the kidney, lung, liver, gastro-intestinal tract, breast, prostate, ovary, and brain. These factors are engaged in brain development, angiogenesis and vascular invasion, wound healing as well as in placenta development and embryogenesis. Plasminogen activators u-PA and t-PA, their inhibitors PAI-1 and PAI-2, and the u-PA-receptor (u-PAR, CD87) are often elevated in solid malignant tumour tissues compared to their normal counterparts. In breast cancer patients, an elevated tumour tissue extract antigen content of u-PA, PAI-1, and u-PAR is associated with increased tumour aggressiveness and poor prognosis; in contrary, an elevated content of t-PA and PAI-2 indicates a favourable prognosis. For clinical relevant determination of these proteolytic factors in tumour tissue extracts, only enzymo-immunometric tests (ELISA) are recommended. Enzymometric and enzymographic tests are actually conducted only in an experimental, preclinical context.

Retinoic acid upregulates the plasminogen activator system in human epidermal keratinocytes.

The activation of the proteolytic plasminogen activator system is important for the re-epithelialization of skin wounds. Keratinocytes synthesize and secrete the urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. Receptor-bound urokinase-type plasminogen activator efficiently activates cell surface bound plasminogen. This results in pericellular proteolysis, which facilitates keratinocyte migration. Urokinase-type plasminogen activator activity is specifically controlled by plasminogen activator inhibitor-1 and -2. As retinoids have been reported to accelerate epithelialization of skin wounds in animal studies and clinical settings, we investigated the effects of all-trans retinoic acid on the plasminogen activator system in human epidermal keratinocytes. As tested in a chromogenic plasminogen activation assay, incubation with 10 microM all-trans retinoic acid caused a marked induction of cell-associated plasminogen activity after 24 h, and this induction was blocked by neutralizing anti-urokinase-type plasminogen activator antibodies, but not anti-tissue-type plasminogen activator antibodies. All-trans retinoic acid lead to a strong increase in urokinase-type plasminogen activator (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) after 24 h. At this time-point, tissue-type plasminogen activator and plasminogen activator inhibitor-1 and -2 proteins were not or only slightly increased. Northern blot analyses revealed that all-trans retinoic acid caused an early and short-lived increase of plasminogen activator inhibitor-1, but a prolonged induction of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA levels. Collectively, these data suggest that all-trans retinoic acid activates the plasminogen activator system in human epidermal keratinocytes by differentially regulating activating and inhibiting components. The activation of the plasminogen activator system may be one mechanism by which all-trans retinoic acid exerts beneficial effects in cutaneous wound healing.

UVB increases urokinase-type plasminogen activator receptor (uPAR) expression.

Keratinocytes synthesize and secrete urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. When bound to urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator proteolytically converts surface bound plasminogen to plasmin, which in turn cleaves many extracellular components leading to pericellular proteolysis. The activation of the urokinase system has been observed during re-epithelialization of skin wounds and in lesions of the autoimmune blistering skin disease pemphigus. As pemphigus is photoinducible, we investigated the effect of ultraviolet B on urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor expression in the epidermal keratinocyte cell line A431. Ultraviolet B increased cellular and secreted urokinase-type plasminogen activator protein (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) 24 h postirradiation. Northern blot analysis indicated that ultraviolet B increased urokinase-type plasminogen activator receptor mRNA. Compared with a more rapid mRNA induction by epidermal growth factor (maximal after 4 h) the ultraviolet B response was maximal after 24 h and prolonged up to 36 h. The mRNA induction was not dependent on protein synthesis as judged by cycloheximide incubation. Ultraviolet B did not influence urokinase-type plasminogen activator receptor mRNA stability (actinomycin D incubation). A transiently transfected chloramphenicol acetyltransferase-reporter construct containing a -398/+51 urokinase-type plasminogen activator receptor promoter fragment was activated when cells were exposed to ultraviolet B. This induction was almost completely abolished by mutating a -182/-176 AP-1 binding sequence. Ultraviolet B increased the binding capacity at this AP-1 motif in electrophoretic mobility shift assays. These data identify a distinct transcriptional mechanism by which ultraviolet B induces urokinase-type plasminogen activator receptor. The epidermal induction of components of the proteolytic urokinase system by ultraviolet B may help explain the photoinducibility of pemphigus lesions.

pYLZ vectors: Saccharomyces cerevisiae/Escherichia coli shuttle plasmids to analyze yeast promoters.

Two yeast/Escherichia coli shuttle vectors have been constructed to analyze promoter structures in Saccharomyces cerevisiae: the multicopy vector, pYLZ-2, and the centromere-based vector, pYLZ-6. Both plasmids contain the coding region of lacZ from E. coli lacking the N-terminal eight amino acids. The truncated reporter gene is preceded by a short polylinker (MCS) suitable for the insertion of promoter fragments. The vectors allow for the study of expression from complete promoters containing UAS and TATA elements, transcriptional start point(s) and the original context of the ATG start codon of a yeast gene. A yeast terminator fragment has been inserted 3' of the lacZ coding region. It contains the transcription termination region of the convergently transcribed yeast genes, GCY1 and PFY1, together with sequences corresponding to the mapped 3'-ends of the respective mRNAs. As an example, reporter activity was measured with promoter fragments from three yeast genes (GCY1, PFY1 and LEO1). The results demonstrate the efficiency of the plasmids for studying constitutive and regulated transcription, both at high and low levels of expression.

Transcriptional control by galactose of a yeast gene encoding a protein homologous to mammalian aldo/keto reductases.

Expression of the S. cerevisiae gene, GCY, encoding a 35-kDa protein with striking homology to mammalian aldo/keto reductases, is under the control of galactose: the intracellular concentration of the respective mRNA (about 1300 nt in length) varies strongly with the carbon source. It is particularly high when galactose is the sole energy source but is low as soon as glucose is present. Lactate, glycerol and raffinose lead to intermediate expression. Both Northern blot analyses and lacZ fusion data indicate a 20- to 50-fold increase in the steady state concentrations of mRNA and beta Gal activity, respectively, when grown on galactose as compared to glucose. The gene is derepressed after cultivation on glycerol in the wt and in a gal80 mutant background but remains uninducible by galactose in strains carrying either a gal2 or a gal4 mutation, affecting galactose permease and the GAL gene trans-activator, respectively. Analysis of GCY expression in gal regulatory mutants reveals epistasis interactions of the gal4 and the gal80 mutations as expected if GCY is regulated by the Gal control system. Repression of GCY transcription by glucose is observed in all three above gal mutant strains. The results suggest that the gene is both positively controlled by galactose and negatively by glucose. Analysis of a set of upstream deletions identifies a single UAS matching the consensus for GAL gene upstream regulation sites. By contrast to other genes regulated by galactose, disruption mutants of GCY exhibit no obvious phenotype, and in particular do not lose the ability to grow on and adapt to galactose. Enzyme tests with AKR-specific substrates suggest that GCY encodes a carbonyl reductase.

The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase.

The gene URA6 encoding uridylate kinase (UK) from Saccharomyces cerevisiae was isolated as a multicopy suppressor of the respiratory-deficient phenotype of an S. cerevisiae mutant defective in the gene AKY2 encoding AMP kinase (AK). The URA6 gene also restored temperature resistance to two different temperature-sensitive mutations in the gene encoding Escherichia coli AK. By contrast, the gene encoding UK of Dictyostelium discoideum on a multicopy yeast shuttle plasmid, expressed under control of the constitutive yeast AKY2 promoter, failed to complement the deficiency in yeast, although such transformants expressed high UK activity. We show that yeast UK exerts significant AK activity which is responsible for the complementation and is absent in the analogous enzyme from D. discoideum. Since UK also significantly phosphorylates CMP (but not GMP), it must be considered an unspecific short-form nucleoside monophosphate kinase. Wild-type mitochondria lack UK activity, but import AKY2. Since multicopy transformation with URA6 heals the Pet- phenotype of AKY2 disruption mutants, the presence of AKY2 in the mitochondrial intermembrane space is not required to maintain respiratory competence. However, furnishing UK with the bipartite intermembrane space-targeting presequence of cytochrome c1 improves the growth rates of AKY2 mutants with nonfermentable substrates, suggesting that AK activity in mitochondria is helpful, though not essential for oxidative growth.

A nuclear yeast gene (GCY) encodes a polypeptide with high homology to a vertebrate eye lens protein.

We describe here the nuclear gene for a yeast protein showing unexpectedly high homology with mammalian aldo/keto reductases as well as with p-crystallin, one of the prominent proteins of the frog eye lens. Although it could be proven that the gene occurs as a single copy in the haploid yeast genome, replacement of the intact by a disrupted, nonfunctional allele led to no obvious phenotype, indicating that the gene is dispensable. The gene was assigned to chromosome XV. It is transcribed in vivo into an mRNA of about 1300 bases with a coding capacity for a protein of 312 amino acids (estimated Mr 35,000).

Determination and mutational analysis of the phosphorylation site in the hypusine-containing protein Hyp2p.

Electrospray mass spectrometry of the purified isoforms of the hypusine-containing protein of Saccharomyces cerevisiae Hyp2p suggested a phosphorylation of the acidic isoform, which was confirmed by phosphatase treatment. The phosphorylation site was mapped to the N-acetylated serine residue in position no. 1 by mass spectrometric analysis of enzymatic fragments. Mutation of this serine residue gives rise to only the basic isoform, confirming our protein chemical data. As this mutation has no effect on cell viability or growth rate, the unphosphorylated isoform is sufficient to exert the essential in vivo function of Hyp2p.

In vivo import of yeast adenylate kinase into mitochondria affected by site-directed mutagenesis.

Site-directed mutagenesis and deletions were used to study mitochondrial import of a major yeast adenylate kinase, Aky2p. This enzyme lacks a cleavable presequence and occurs in active and apparently unprocessed form both in mitochondria and cytoplasm. Mutations were applied to regions known to be surface-exposed and to diverge between short and long isoforms. In vertebrates, short adenylate kinase isozymes occur exclusively in the cytoplasm, whereas long versions of the enzyme have mitochondrial locations. Mutations in the extra loop of the yeast (long-form) enzyme did not affect mitochondrial import of the protein, whereas variants altered in the central, N- or C-terminal parts frequently displayed increased or, in the case of a deletion of the 8 N-terminal triplets, decreased import efficiencies. Although the N-terminus is important for targeting adenylate kinase to mitochondria, other parameters like internal sequence determinants and folding velocity of the nascent protein may also play a role.

High levels of profilin suppress the lethality caused by overproduction of actin in yeast cells.

Overproduction of actin is lethal to yeast cells. In contrast, overexpression of the profilin gene, PFY1, encoding an actin-binding protein, leads to no very obvious phenotype. Interestingly, profilin overproduction can compensate for the deleterious effects of too much actin in a profilin concentration-dependent manner. Our results, thus, document that actin and profilin interact in vivo. Immunofluorescence studies suggest that suppression works by reducing actin assembly. We observed, however, that even massive overproduction of profilin fails to fully restore the wild-type phenotype (e.g. the wild-type appearance of the actin microfilament system). This may indicate that actin monomer sequestration is not the only mechanism by which the balance of actin polymerization is controlled.

Yeast adenylate kinase is transcribed constitutively from a promoter in the short intergenic region to the histone H2A-1 gene.

Yeast mitochondrial adenylate kinase (high molecular mass form, gene locus: AKY2) is encoded on chromosome IV of the same DNA strand as histone H2A-1. The nontranslated intergenic region spans 560 bp, the nontranscribed spacer can be estimated to comprise at most 300 bp. The TATA-box sequence is contained in a striking environment consisting of 20 alternating pyrimidines and purines. The AKY2 transcript is made constitutively: (i) the cellular mRNA concentration does not vary significantly with either growth conditions or elapse of the cell cycle; (ii) beta-galactosidase activity is about constant in yeast cells grown on various carbon sources after transformation with AKY2-promoter/lacZ fusions; (iii) primer elongation analysis shows that utilization of 5 initiation sites is qualitatively and quantitatively independent of the growth conditions and the carbon source used; (iv) Western blot analysis and adenylate kinase activity measurements indicate the absence of post-transcriptional controls as well.