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Biomedical subjects

E Koivunen

Publications and source records attributed to E Koivunen.

At least 37 records · Page 2Linked to original sources
PubMed2000

Development of novel peptide ligands modulating the enzyme activity of prostate-specific antigen.

Prostate-specific antigen (PSA) is a serine proteinase produced mainly by epithelial cells of the prostate. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. The major problem of the PSA determination in early diagnosis is the high false positive rate due to benign prostatic hyperplasia, but the clinical accuracy can be improved by determining the proportions of various molecular forms of PSA. The main biological function of PSA is liquefaction of the seminal gel formed after ejaculation, but PSA has also been suggested to regulate invasiveness and metastatic potential of prostatic tumors. Thus, agents binding to and affecting the function of PSA have the potential to be used for diagnosis and therapy of prostate cancer. We have developed peptides specific for PSA by using cyclic phage display peptide libraries. After deducing the amino acid sequence of the peptides by sequencing the relevant part of phage genome, the peptides were expressed as glutathione-S-transferase (GST) fusion proteins or produced by chemical synthesis. The peptides were shown to bind to PSA specifically as indicated by lack of binding to other related serine proteinases. The binding of the peptides with PSA was strongly inhibited by monoclonal antibodies specific for free PSA and they did not bind to PSA-inhibitor complexes indicating that they bind close to the active site of the enzyme. Most of the peptides enhanced the enzyme activity of PSA against a chromogenic substrate. The affinity of the peptides could be increased by including Zn2+ in the reaction mixture. These results show that peptides that bind to PSA and modulate its enzyme activity can be developed by phage display techniques. These peptides have the potential to be used for targeting of prostatic tumors and diagnostics of prostate cancer.

Humans↗
PubMed1999

Phage-displayed peptides mimicking the discontinuous neutralization sites of puumala Hantavirus envelope glycoproteins.

We selected peptide ligands mimicking the surface structure of discontinuous binding sites of Puumala hantavirus-neutralizing monoclonal antibodies from a random 18-amino acid peptide library containing a disulfide bridge in a fixed position and displayed on a filamentous phage. The varying of selection conditions, either by shortening of the association time or by competitive elution with antigen, was crucial for the selection of peptide inserts that could be aligned with the primary sequences of the envelope glycoproteins G1 and G2. Correspondingly, when the envelope glycoprotein sequences were synthesized as overlapping peptides as spots on membrane, the same site in primary structure was found as with phage display, which corroborates the use of the two methods in mapping of conformational epitopes. Also, epitopes reactive with early-phase sera from Puumala virus infection were defined with the pepspot assay in the amino-terminal region of G1. Similarities of the selected phage clones to a monoclonal antibody-escape mutant site and to a linear early-phase epitope were found.

Amino Acid Sequence↗
PubMed1999

Tumor targeting with a selective gelatinase inhibitor.

Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.

Amino Acid Sequence↗
PubMed1999

Response level and survival after conventional chemotherapy for multiple myeloma: a Finnish Leukaemia Group study.

The relation between overall survival time (OS) and response level (CR, 75%R, PR, MR, SD) was analysed in 432 multiple myeloma patients from 4 prospective Finnish Leukaemia group trials, treated with conventional chemotherapy. The primary regimen was either melphalan and prednisolone or combination chemotherapy with melphalan as a main component. Both the influence of the pre-treatment factors in patients with different levels of responses and the aspects related to chemotherapy were analysed. The 324 patients aged up to 70 yr and the 10(8) older patients were dealt with as separate groups. Irrespective of the primary chemotherapy regimen, the level of response was not significantly influencing in the OS time, and this was true in both age categories. The median OS of the patients up to 70 yr of age who had any response was 57 months, compared to the 10 months of those with PD (p < 0.001). The corresponding figures for the older patients were 40 and 4 months (p <0.001), respectively. The pretreatment prognosticators for the patients with minimal responses were not more favourable than for patients with responses at higher levels. The prolonged primary chemotherapy was favourable in patients having responses less than PR. Accordingly, the primary goal of conventional chemotherapy for multiple myeloma is stabilization of disease, not the level of response.

Aged↗
PubMed1999

Response rate and survival after conventional chemotherapy for multiple myeloma by hospitals with different inclusion rates of patients to the trials. A Finnish Leukemia Group study.

The purpose of this study was to analyse the influence of the hospital size, measured as the number of annual patient enrolments in the Finnish Leukaemia Group trials in the period 1979-85, on response rate and survival after conventional chemotherapy for multiple myeloma. The 432 myeloma patients for this study were derived from 4 prospective multicentre trials of the Finnish Leukaemia Group. The comparisons of the response rate to primary chemotherapy, progression-free survival time (PFS), response rate after first relapse and overall survival time (OS) were made between patients treated in 3 hospital categories according to annual enrolment rate. 273 of the patients had been treated in "large" hospitals, 120 in "intermediate" and 38 in "small" ones. The median OS of patients aged up to 70 yr was 49 months and that of patients older than 70 yr was 31 months. There were no significant differences in response rate, PFS, response rate after first relapse and OS between the hospital categories. The most important endpoints in the treatment of multiple myeloma, response rate, PFS and OS are independent of the size of the hospital, measured as the number of annual patient entries in the trial. This allows the decentralisation of the conventional chemotherapy of multiple myeloma, within the framework of properly organised clinical trials.

Aged↗
PubMed1999

Identification of receptor ligands with phage display peptide libraries.

With the development and maturation of the technology of displaying peptides on bacteriophage, it has become possible to isolate peptide ligands to various targets. In the phage display strategy, up to 10(9) peptides of different permutations are expressed on the surface of filamentous phage. Thus, peptides capable of binding target molecules in vitro and even target tissues in vivo can be identified. In recent years, a series of libraries that display degenerate peptides of different lengths have been constructed, and specific ligands to cell surface receptors, such as integrins, have been isolated. In the in vivo biopanning, peptides targeting distinct organs or tumors have been rescued after intravenous administration of phage libraries into mice. In one application, the isolated peptide ligands have been used to direct a cytotoxic drug to tumor vasculature in mice. Further applications in radioimaging and radiotherapy are being investigated.

Animals↗
PubMed1998

Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display.

Vascular beds are known to differ in structure and metabolic function, but less is known about their molecular diversity. We have studied organ-specific molecular differences of the endothelium in various tissues by using in vivo screening of peptide libraries expressed on the surface of a bacteriophage. We report here that targeting of a large number of tissues with this method yielded, in each case, phage that homed selectively to the targeted organ. Different peptide motifs were recovered from each of these tissues. The enrichment in homing to the target organs relative to an unselected phage was 3-35-fold. Peptide sequences that conferred selective phage homing to the vasculature of lung, skin, and pancreas were characterized in detail. Immunohistochemistry showed that the phage localized in the blood vessels of their target organ. When tested, the phage homing was blocked in the presence of the cognate peptide. By targeting several tissues and by showing that specific homing could be achieved in each case, we provide evidence that organ- and tissue-specific molecular heterogeneity of the vasculature is a general, perhaps even universal, phenomenon. Our results also show that these molecular differences can serve as molecular addresses.

Animals↗
PubMed1998

Comparison between four and eight cycles of intensive chemotherapy in adult acute myeloid leukemia: a randomized trial of the Finnish Leukemia Group.

In acute myelogenous leukemia (AML) intensive postremission treatment is needed for an optimal result. However, it is not known how long the treatment should last and how many courses are necessary. The object of this prospective study was to compare four and eight intensive chemotherapy cycles in the treatment of adult de novo AML. In a multicenter study, 248 consecutive patients, aged from 16 to 65 years, were treated with intensive induction treatment. The patients in remission after two courses were randomized to receive either two (short arm) or six (long arm) additional intensive cycles of chemotherapy. The median follow-up time of the living patients is 68 months. Of the patients, 77% achieved complete remission, and 36% of all patients survived for 5 years. Seventy-three patients were randomized to the short arm and 66 to the long arm. There was no significant difference in the relapse-free survival (median 21 months vs 17 months) or overall survival (43 months vs 39 months) between the short and long arms, respectively. Treatment-related deaths occurred in 31 patients (13%), 11 of them in first remission. More than one-third of the patients survived for 5 years. It seems probable that the first few months after diagnosis are decisive for the prognosis if the chemotherapy is intensive, and further treatment cannot markedly influence the outcome.

Aclarubicin↗
PubMed1998

Successful treatment of platelet transfusion refractoriness: the use of platelet transfusions matched for both human leucocyte antigens (HLA) and human platelet alloantigens (HPA) in alloimmunized patients with leukaemia.

Six patients, 4 with acute myeloid leukaemia and 2 with a myelodysplastic syndrome who were refractory to random donor platelet transfusions and alloimmunized to human leucocyte antigens (HLA) and human platelet alloantigens (HPA), were treated with HLA- and HPA-matched platelet transfusions. In all the patients refractoriness and alloantibodies to HLA as well as HPA-1b or HPA-5b were detected simultaneously. Sixty-seven transfusions (445 units) of HLA- and HPA-matched platelets were given and responses to them were, in general, satisfactory in all the patients. No major spontaneous bleeding occurred. Four patients underwent bone marrow transplantation despite alloimmunization. The percentages of platelet transfusion days with a platelet nadir below 20x10(9)/l were 88% for the last 3 random donor platelet transfusions and 39% for the first 3 HLA- and HPA-matched platelet transfusions, respectively (p=0.009, Fisher's exact test). Four patients received also HLA-matched platelets, but responses to them were poor. The small number of transfusions with HLA-matched platelets precluded comparisons to either the random donor or HLA- and HPA-matched platelet transfusions. It seems that HLA- and HPA-alloimmunized patients can be successfully supported with HLA- and HPA-matched platelet concentrates.

Adult↗
PubMed1997

Activation of type IV procollagenases by human tumor-associated trypsin-2.

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62-65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.

Amino Acid Sequence↗
PubMed1997

Cell-surface interactions of echovirus 22.

Echovirus 22 (EV22) is a picornavirus forming a distinct molecular cluster together with echovirus 23. EV22 has an Arg-Gly-Asp (RGD) peptide motif in its capsid protein VP1; similar motifs are known to mediate many cell-cell and microbe-host interactions. To identify peptide sequences that specifically bind to EV22 and potentially play a role in receptor recognition, we have used here peptide libraries displayed in filamentous phage. We isolated an EV22-binding motif CLRSG(R/F)GC. The synthetic CLRSGRGC peptide was able to inhibit EV22 infection. The infection was also inhibited by an RGD-containing peptide representing the C terminus of the EV22 capsid protein VP1 and CWDDGWLC (an RGD-binding peptide; Pasqualini, R., Koivunen, E., and Ruoslahti, E. (1995) J. Cell Biol. 130, 1189-1196). As the EV22-recognizing sequence LRSG is found in the integrin beta1 chain and the entire LRSGRG hexapeptide occurs in the matrix metalloproteinase 9 (MMP-9), we carried out blocking experiments with anti-integrin and anti-MMP-9 antibodies. EV22 infection could be blocked in cell cultures with anti-alphav, -beta1, and, to a lesser extent, with anti-MMP-9 antibodies. These results imply that EV22 recognizes preferentially alphavbeta1-integrin as a cellular receptor and MMP-9 may also play a role in the cell-surface interactions of the virus.

Amino Acid Sequence↗
PubMed1997

Intensive chemotherapy of poor prognosis myelodysplastic syndromes (MDS) and acute myeloid leukemia following MDS with idarubicin and cytarabine.

Forty patients with high risk myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) preceded by MDS were treated with intensive induction and consolidation chemotherapy in a prospective multicenter pilot study. They were given two cycles of cytarabine 100 mg/m with 12-h intervals on days 1-7 and idarubicin 12 mg/m2 on days 5-7, both intravenously. Patients who were in remission after these two cycles were given two further cycles of cytarabine on days 1-5 and idarubicin on day 5. No maintenance treatment was given. Eleven out of 19 MDS patients (58%) and 10 out of 21 AML patients (48%), in total 21 out of 40 patients (53%), entered remission. Eight patients underwent allogeneic bone marrow transplantation. The follow-up time was 13-48 (median 33) months. At the time of the analysis, seven patients survived, four patients with MDS all of whom had been treated with bone marrow transplantation (three in continuous remission), and three patients with AML treated with chemotherapy only (two in continuous remission). The median survival of the patients treated with chemotherapy only was 12 months, with the median progression-free survival being 8 months. In view of the poor prognostic factors of the patients, the remission rate was satisfactory, but the responses as well as the survival were short. The post-remission treatment needs to be improved.

Adult↗
PubMed1997

Alpha v integrins as receptors for tumor targeting by circulating ligands.

Phage displaying an Arg-Gly-Asp (RGD)-containing peptide with a high affinity for alpha v integrins homed to tumors when injected intravenously into tumor-bearing mice. A substantially higher amount of alpha v-directed RGD phage than control phage was recovered from malignant melanomas and breast carcinoma. Antibodies detected the alpha v-directed RGD phage in tumor blood vessels, but not in several normal tissues. These results show that the alpha v integrins present in tumor blood vessels can bind circulating ligands and that RGD peptides selective for these integrins may be suitable tools in tumor targeting for diagnostic and therapeutic purposes.

Amino Acid Sequence↗
PubMed1996

Serum samples from pancreatectomized patients contain trypsinogen immunoreactivity.

The concentrations of trypsinogen-1 and -2 in serum samples from patients who have undergone pancreatectomy were measured by highly sensitive and specific time-resolved immunofluorometric assays. The isoenzyme pattern was determined by ion-exchange chromatography and determination of immunoreactivity in the fractions. All samples contained trypsinogen-2, the mean level being one fifth of that in healthy controls. Trypsinogen-1 was detected in one of nine samples. In addition to the main trypsinogen isoenzymes, we observed in normal serum two trypsinogen isoenzymes previously found in mucinous ovarian cyst fluid. Our results suggest that trypsinogen is not exclusively expressed by the pancreas and certain tumors but that it also may be produced by normal extrapancreatic tissues. This should be considered when an assay of trypsinogen in serum is used for clinical purposes.

Adenocarcinoma↗
PubMed1996

A polymeric form of fibronectin has antimetastatic effects against multiple tumor types.

Metastasis accounts for most deaths in cancer patients. Tumor cell adhesion to the extracellular matrix through integrins is thought to be a critical step in metastasis and a potential target for therapeutic intervention. We show here that treatment of human osteosarcoma, melanoma and carcinoma cells with a polymeric form of fibronectin (sFN), before inoculation into nude mice, prevented tumor formation. Intraperitoneally administered sFN significantly reduced lung colonization from intravenously injected tumor cells (experimental metastasis) and from subcutaneous tumors in nude mice (spontaneous metastasis). Treatment with sFN blocked cell spreading and migration in vitro suggesting a possible mechanism for the antimetastatic effect.

Animals↗
PubMed1996

Peptides in cell adhesion: powerful tools for the study of integrin-ligand interactions.

Many of the cell adhesion receptors recognize simple sequences that can be reproduced as synthetic peptides. This circumstance has led to a widespread use of peptides as modulators of cell adhesion. Peptides capable of binding to cell adhesion receptors, such as the integrins, can be used as mediators of cell attachment to a surface by coating the surface with the peptide. Peptides bound to a solid phase can also be used to isolate adhesion receptors by affinity chromatography. Alternatively, the peptides can be used to inhibit cell attachment to the natural ligands of the adhesion receptors. In either mode, adhesion peptides have proven to be highly useful probes in cell adhesion research and they also show promise as a new class of therapeutics. The purpose of this article is to review some of the properties and uses of adhesion peptides.

Binding Sites↗
PubMed1995

A novel matrix attachment region DNA binding motif identified using a random phage peptide library.

SATB1 is a nuclear matrix attachment DNA (MAR)-binding protein which is predominantly expressed in thymocytes. This protein binds to the minor groove specifically recognizing an unusual DNA context exhibited by a specific MAR region with strong base-unpairing propensity. A phage library displaying nonamer random peptides without any built-in structure was used to identify a MAR binding motif of SATB1. One predominant cyclic peptide C1 of CRQNWGLEGC selected by a MAR-affinity column showed 50% identity with a segment in SATB1 (amino acids 355-363). Replacement of the C1 similarity segment in SATB1 by a random amino acid sequence or its truncation resulted in more than 80% reduction in MAR binding. In contrast, replacement of the same SATB1 segment with the C1 peptide restored full MAR binding activity and specificity as the wild-type protein. Single amino acid mutation of the conserved Arg or Glu residue to Ala greatly reduced MAR binding. Taken together our data show that a nine amino acid sequence in SATB1 represents a key MAR binding motif. Phage display may provide a general tool for rapid identification of DNA binding peptide motifs.

Amino Acid Sequence↗