Markku Kallajoki

Apoptosis in Human Acute Myocardial Infarction

BACKGROUND After reopening of the infarct-related coronary artery, cardiomyocytes continue to die during reperfusion. The mechanisms of cell death have been subject to debate. We studied whether an apoptotic type of cell death occurs in human acute myocardial infarction (AMI). METHODS AND RESULTS We studied myocardial samples of eight patients who died of AMI and had patent infarct-related arteries at autopsy. Six of the patients had received initially successful thrombolysis. Extensive formation of DNA strand breaks, the typical biochemical feature of apoptosis, was detected with the use of the in situ DNA end-labeling method. Apoptotic cardiomyocytes were observed particularly in the border zones of histologically infarcted myocardium, whereas very few apoptotic cells were present in the remote noninfarcted myocardium. Internucleosomal fragmentation was confirmed by agarose gel electrophoresis of DNA isolated from the representative myocardial areas. CONCLUSIONS This study provides evidence that in addition to overt necrosis, a subset of myocytes undergo apoptosis during ischemia-reperfusion injury. Apoptosis may provide a new target for cardioprotection during evolving AMI in humans.

Transient coexpression of cytokeratin and vimentin in differentiating rat sertoli cells

The expression of cytokeratin and vimentin type intermediate filaments were studied in fetal, postnatal, and adult rat testes. Immunocytochemical observations were correlated with the light and electron microscopic analysis of the developing organs. The Sertoli cell precursors in 15-day-old fetal testes contained both cytokeratin and vimentin. A gradual reorganization of both filaments, accompanied by a decrease of cytokeratin-positivity, was observed toward the end of the fetal period. The simultaneous presence of cytokeratin and vimentin in the same cells was shown by double immunofluorescence of newborn testes and the primary culture of dissociated testicular cells. In postnatal Sertoli cells, cytokeratin-positivity continued to decrease and disappeared by the age of 14 days. The increase in vimentin content and the appearance of axially oriented vimentin filaments coincided with the acquisition of the columnar shape of the Sertoli cells. The presence of cytokeratin and vimentin in fetal and newborn testes, and only vimentin in the adult testes was confirmed by immunoblotting. The present results suggest that major qualitative changes in the expression of intermediate filament proteins can take place during the embryonic development. The expression of cytokeratin in developing Sertoli cells, although only transient, supports the epithelial origin of these cells and can be applied as a marker for embryonic and early postnatal Sertoli cells.

Gastrointestinal stromal tumors with internal tandem duplications in 3' end of KIT juxtamembrane domain occur predominantly in stomach and generally seem to have a favorable course.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. GISTs express KIT and have KIT mutations. Majority of these mutations cluster in the 5′ end of the KIT juxtamembrane domain. Little is known about the clinicopathological profile of GIST carrying internal tandem duplications in the 3′ end of KIT juxtamembrane domain (ITDs in the 3′ KIT-JM). In this study, 500 immunohistochemically KIT-positive GISTs were screened for this type of mutation, and 18 cases were identified (3.6%). The majority of the ITDs consisted of 1 to 18 codon duplications, with Tyr578, Asp579, and Leu576 being the most commonly duplicated codons. There were 14 gastric (78%), 2 small intestinal (11%), and 2 anal (11%) primary tumors diagnosed in 12 females and 6 males with median age of 71 years. The frequency of IDTs in gastric GISTs was 6.5% and was only 0.5% in intestinal GISTs. There was a strong female predominance (79%) among the patients with gastric tumors. Histologically, 16 GISTs were spindle cell, and 2 had epithelioid morphology. The sizes of primary tumors varied from 1 to >20 cm. Based on the combination of tumor size and mitotic activity, six tumors were classified as benign or probably benign, eight as having uncertain malignant potential, and only four as malignant. Follow-up data available in 17 patients confirmed the malignant course of disease in 3 cases. Only one of the tumors classified as potentially malignant metastasized, although the follow-up was limited in some cases. In summary, the great majority of GISTs with ITDs in the 3′ KIT-JM were mitotically inactive tumors occurring predominantly in the stomach and that seemed to have a favorable course. This suggests that presence of these IDTs may define a clinicopathologically favorable subset of GISTs. The consequence of these mutations to KIT signaling should be investigated.

p38α and p38δ mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells

Recent studies indicate that the specificity of p38 mitogen-activated protein kinase (MAPK)-mediated cellular stress responses is determined by the expression pattern of the distinct p38 isoforms. Here, we have analysed the function of distinct p38 isoforms in the growth and invasion of head and neck squamous cell carcinomas (HNSCCs). Activation of p38 MAPK by arsenite resulted in inactivation of the ERK1,2 signaling pathway by dephosphorylation of MEK1,2 in primary human epidermal keratinocytes (HEKs), whereas in HNSCC cells this p38-mediated inhibition of the ERK1,2 pathway was absent. Quantitation of p38 pathway component mRNA expression in HNSCC cell lines (n=42) compared to HEKs (n=8) revealed that p38α and p38δ isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38α or p38δ activity by adenoviral delivery of corresponding dominant-negative p38 isoforms potently reduced MMP-13 and MMP-1 expressions, and suppressed the invasion of HNSCC cells through collagen. Dominant-negative p38α and p38δ inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38α activity also compromised survival of SCC cells. p38α and p38δ were predominantly expressed in HNSCCs (n=24) and nonneoplastic epithelium in vivo (n=6), with MKK3 being the primary upstream activator. Activation and expression of p38α and p38δ by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38α or p38δ in cutaneous SCC cells potently inhibited their implantation in skin of severe combined immunodeficiency mice and growth of xenografts in vivo. Our results indicate that p38α and p38δ specifically promote the malignant phenotype of SCC cells by regulating cell survival, proliferation and invasion, suggesting these p38 MAPK isoforms as potential therapeutic targets in HNSCCs.

In Vivo Imaging of Prostate Cancer Using [68Ga]-Labeled Bombesin Analog BAY86-7548

Purpose: A novel [68Ga]-labeled DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 peptide (BAY86-7548) having high affinity to bombesin receptor subtype II to detect primary and metastatic prostate carcinoma using positron emission tomography/computed tomography (PET/CT) was synthesized and evaluated for prostate cancer. Experimental Design: In this first human study with BAY86-7548, 14 men scheduled for radical prostatectomy (n = 11) or with biochemical recurrence after surgery or hormonal therapy (n = 3) were enrolled. The patients received an intravenous injection of BAY86-7548 followed by over 60-minute dynamic imaging of prostate gland (n = 10) and/or subsequent whole-body imaging (n = 14). The visual assessment of PET/CT images included evaluation of intraprostatic (12 subsextants) and pelvic nodal uptake of BAY86-7548 in 11 surgical patients and detection of potential metastatic foci in all patients. In patients with biochemical recurrence, results were compared with those of either [11C]-acetate (n = 2) or [18F]-fluoromethylcholine (n = 1) PET/CT. Results: We found a sensitivity, specificity, and accuracy of 88%, 81% and 83%, respectively, for detection of primary PCa and sensitivity of 70% for metastatic lymph nodes using histology as gold standard. BAY86-7548 correctly detected local recurrence in prostate bed and showed nodal relapse in accordance with [11C]-acetate PET/CT in 2 patients with biochemical relapse. In the third hormone refractory patient, BAY86-7548 failed to show multiple bone metastases evident on [18F]-fluoromethylcholine PET/CT. Conclusion: BAY86-7548 PET/CT is a promising molecular imaging technique for detecting intraprostatic prostate cancer. Clin Cancer Res; 19(19); 5434–43. ©2013 AACR.

Vascular adhesion protein-1, intercellular adhesion molecule-1 and P-Selectin mediate leukocyte binding to ischemic heart in humans

OBJECTIVES The expression of endothelial adhesion molecules and their functional significance in leukocyte adhesion to human myocardial blood vessels in acute myocardial infarction (AMI) were studied. BACKGROUND Leukocyte extravasation, mediated by specific adhesion molecules, exacerbates tissue injury after restoration of blood supply to an ischemic tissue. Experimental myocardial reperfusion injury can be alleviated with antibodies that block the function of adhesion molecules involved in leukocyte emigration, but the relevant molecules remain poorly characterized in human AMI. METHODS Semiquantitative immunohistochemistry and in vitro adhesion assays were used to study the expression and granulocyte binding abilities of different endothelial adhesion molecules in human AMI. Changes in the molecular nature of vascular adhesion protein-1 (VAP-1) were evaluated using immunoblotting. RESULTS Certain endothelial adhesion molecules (intercellular adhesion molecule [ICAM-2], CD31 and CD73) were expressed in myocardial blood vessels homogeneously in normal and ischemic hearts, whereas others (E-selectin and peripheral lymph node addressin) were completely absent from all specimens. The synthesis of ICAM-1 was locally, and that of P-selectin regionally, upregulated in the infarcted hearts when compared with nonischemic controls. Vascular adhesion protein-1 showed ventricular preponderance in expression and alterations in posttranslational modifications during ischemia-reperfusion. Importantly, P-selectin, ICAM-1 and VAP-1 mediated granulocyte binding to blood vessels in the ischemic human heart. CONCLUSIONS Human P-selectin, ICAM-1 and VAP-1 appear to be the most promising targets when antiadhesive interventions preventing leukocyte-mediated tissue destruction after myocardial ischemia are planned.

Cardiomyocyte apoptosis in experimental coxsackievirus B3 myocarditis

INTRODUCTION Viruses are known to induce apoptosis in their host cells. We studied whether cardiomyocyte apoptosis occurs upon coxsackievirus B3 (CVB3) infection and whether virus-associated apoptosis plays a role in the pathogenesis of experimental myocarditis. METHODS BALB/c mice were infected with two variants of CVB3 causing either mild or severe myocarditis. Myocardial and serum samples were collected from Day 1 to Day 14 after virus inoculation. Apoptosis was detected in myocardial tissue sections using the terminal transferase-mediated DNA nick end labelling (TUNEL) assay and staining of active caspase 3, and compared with the presence of infectious CVB3 and viral proteins in cardiomyocytes. RESULTS Compared with the noninfected control mice, infection with either CVB3 variant resulted in significantly increased cardiomyocyte apoptosis, which peaked on Day 5 after infection. At this time, the average percentages of apoptotic cardiomyocytes were 0.17% (SD 0.04; P=.03) and 0.77% (SD 0.11; P<.01) in mild and severe disease forms, respectively. The amount of apoptosis correlated with titers of infectious CVB3 in the heart muscle. Viral proteins were detected in the TUNEL-positive cells by immunohistochemistry. In the late stages of disease, apoptosis, together with inflammatory infiltrates persisted only in the severe disease form. CONCLUSIONS CVB3-associated myocardial damage involves cardiomyocyte apoptosis. In the early stages of the disease, it appears to be triggered by viral replication in the cardiomyocytes.

Targeted inhibition of human collagenase-3 (MMP-13) expression inhibits squamous cell carcinoma growth in vivo.

Squamous cell carcinomas (SCCs) of the head and neck are characterized by a high tendency for local invasion and metastasis to lymph nodes. Collagenase-3 (MMP-13) is specifically expressed by tumor cells in SCCs of the head and neck and its expression correlates with their invasion capacity. To specifically examine the role of MMP-13 in the growth and invasion of SCC, we constructed a hammerhead ribozyme targeted against human MMP-13 mRNA. The anti-MMP-13 ribozyme effectively cleaved MMP-13 transcripts in vitro. Adenoviral delivery of the anti-MMP-13 ribozyme to cutaneous metastatic SCC cells in culture resulted in potent and specific inhibition of the production of proMMP-13 and markedly suppressed invasion of SCC cells through Matrigel. In addition, adenoviral delivery of anti-MMP-13 ribozyme promoted apoptosis in SCC cells within 72 h. Intratumoral injection of anti-MMP-13 ribozyme coding adenovirus into human SCC xenografts established in SCID mice potently suppressed tumor growth, inhibited MMP-13 expression and gelatinolytic activity and reduced the number of proliferating cells within the tumors. These results provide evidence for an important role for MMP-13 in SCC growth and invasion and identify MMP-13 as a promising target for ribozyme-based therapy of SCC in vivo.

Oncolytic Capacity of Attenuated Replicative Semliki Forest Virus in Human Melanoma Xenografts in Severe Combined Immunodeficient Mice

Oncolytic viruses have gained attention as a novel form of cancer treatment. Many viral vectors in use today have been rendered safe by deletion of genes encoding viral structural proteins, thus making them unable to spread beyond the first infected cells. Hence, such replication-deficient constructs may lack efficacy. Here, we analyzed the oncolytic potential of the replication-competent vector VA7-EGFP, based on the avirulent Semliki Forest virus (SFV) strain A7(74), to kill cancer cells in culture as well as to target s.c. human melanoma xenografts in severe combined immunodeficient (SCID) mice. VA7-EGFP was able to infect most cancer cell lines studied, leading to complete lysis of the cells within 72 hours after infection. In SCID mice grafted with A2058 human melanoma, marked regression of the xenografts was observed following a single injection of 10(6) plaque-forming units of virus given either i.p., i.v., or intratumorally. Histologic analysis revealed the presence of virus not only in all treated tumors but also in the brains of the treated mice, causing progressing neuropathology beginning at day 16 after infection. Following initial oncolysis, clusters of viable tumor cells were observed embedded in connective tissue, and at later stages, encapsulated tumor nodules had formed. Infection of melanoma cells from explant cultures of these nodules revealed that a portion of the cells were resistant to virus. To be eligible for use in virotherapy, the ability of avirulent SFV to spread within tumor tissue may have to be improved and the biological safety of the virus may have to be addressed thoroughly in higher animals.

Matrix metalloproteinase-7 activates heparin-binding epidermal growth factor-like growth factor in cutaneous squamous cell carcinoma

Background  Tumour‐specific expression of matrix metalloproteinase (MMP)‐7 has been noted in cutaneous squamous cell carcinomas (SCCs) in patients with recessive dystrophic epidermolysis bullosa (RDEB).