Tumour-cell-derived complement components C1r and C1s promote growth of cutaneous squamous cell carcinoma.

BACKGROUND: The incidence of epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is increasing worldwide. OBJECTIVES: To study the role of the complement classical pathway components C1q, C1r and C1s in the progression of cSCC. METHODS: The mRNA levels of C1Q subunits and C1R and C1S in cSCC cell lines, normal human epidermal keratinocytes, cSCC tumours in vivo and normal skin were analysed with quantitative real-time polymerase chain reaction. The production of C1r and C1s was determined with Western blotting. The expression of C1r and C1s in tissue samples in vivo was analysed with immunohistochemistry and further investigated in human cSCC xenografts by knocking down C1r and C1s. RESULTS: Significantly elevated C1R and C1S mRNA levels and production of C1r and C1s were detected in cSCC cells, compared with normal human epidermal keratinocytes. The mRNA levels of C1R and C1S were markedly elevated in cSCC tumours in vivo compared with normal skin. Abundant expression of C1r and C1s by tumour cells was detected in invasive sporadic cSCCs and recessive dystrophic epidermolysis bullosa-associated cSCCs, whereas the expression of C1r and C1s was lower in cSCC in situ, actinic keratosis and normal skin. Knockdown of C1r and C1s expression in cSCC cells inhibited activation of extracellular signal-related kinase 1/2 and Akt, promoted apoptosis of cSCC cells and significantly suppressed growth and vascularization of human cSCC xenograft tumours in vivo. CONCLUSIONS: These results provide evidence for the role of tumour-cell-derived C1r and C1s in the progression of cSCC and identify them as biomarkers and putative therapeutic targets in cSCC. What's already known about this topic? The incidences of actinic keratosis, cutaneous squamous cell carcinoma (cSCC) in situ and invasive cSCC are increasing globally. Few specific biomarkers for progression of cSCC have been identified, and no biological markers are in clinical use to predict the aggressiveness of actinic keratosis, cSCC in situ and invasive cSCC. What does this study add? Our results provide novel evidence for the role of complement classical pathway components C1r and C1s in the progression of cSCC. What is the translational message? Our results identify complement classical pathway components C1r and C1s as biomarkers and putative therapeutic targets in cSCC.

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). OBJECTIVES: To examine the potential role of MMP-7 in shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in RDEB-associated and sporadic SCCs. METHODS: Tissue microarrays of RDEB-associated SCC (n = 20), non-EB SCC (n = 60) and Bowen disease (n = 28) were immunostained for MMP-7, CD44 variant 3 (CD44v3) and HB-EGF. Shedding of HB-EGF was studied in vitro using two cutaneous SCC cell lines. RESULTS: Immunohistochemical analysis showed that HB-EGF was absent in tumour cells when MMP-7 and CD44v3 colocalized, and that the absence of HB-EGF was more pronounced in RDEB-associated SCCs than in non-EB SCCs. The loss of HB-EGF in MMP-7-CD44v3 double-positive areas was interpreted to indicate shedding and activation of HB-EGF; this was also detected in Bowen disease indicating its importance in the early phase of SCC development. Specific knockdown of MMP-7 expression in human cutaneous SCC cells by small interfering RNA inhibited shedding of HB-EGF and resulted in diminished activation of the EGF receptor (EGFR) and ERK1/2, and in reduced proliferation of SCC cells. CONCLUSIONS: These findings provide evidence for the role of MMP-7 in promoting the growth of cutaneous SCCs by shedding HB-EGF, and identify EGFR signalling as a potential therapeutic target in RDEB-associated SCC and unresectable sporadic cutaneous SCC.

p38alpha and p38delta 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 p38alpha and p38delta isoforms are predominantly expressed in both cell types and that MKK3 is the primary upstream activator expressed. Inhibition of endogenous p38alpha or p38delta 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 p38alpha and p38delta inhibited squamous cell carcinoma (SCC) cell proliferation and inhibition of p38alpha activity also compromised survival of SCC cells. p38alpha and p38delta 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 p38alpha and p38delta by tumor cells was detected in HNSCCs in vivo (n=16). Adenoviral expression of dominant-negative p38alpha or p38delta 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 p38alpha and p38delta 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.

Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis.

Despite well-documented cardiotoxic effects, doxorubicin remains a major anticancer agent. To study the role of myocardial apoptosis following doxorubicin administration, male Wistar rats were exposed to 1.25, 2.5, and 5 mg/kg of i.p. doxorubicin and terminated on days 1-7 in groups of five. Doxorubicin caused a significant (P < 0.001) and dose-dependent induction of cardiomyocyte apoptosis at 24-48 h after the injection. Repeated injections of 2.5 mg/kg given every other day resulted in peaks of apoptosis at 24 h after each injection. However, no additive effect of repeated dosing was noted. In histological samples, alterations in the cytoskeletal apparatus with focal loss of contractile elements were seen after a single injection. Myocyte necrosis was absent. Thus, acute doxorubicin-induced cardiotoxicity involves cardiomyocyte apoptosis, a potentially preventable form of myocardial tissue loss.

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.

Synthesis of group II phospholipase A2 and lysozyme in lacrimal glands.

PURPOSE: The aim of this study was to demonstrate the synthesis and cellular distribution of group II phospholipase A2 and lysozyme in the main and accessory lacrimal glands. METHODS: The authors studied samples of normal main lacrimal glands of seven autopsied subjects and accessory lacrimal glands of eight patients who underwent ptosis surgery. The specimens were immunostained with a rabbit antiserum against group II phospholipase A2 and a monoclonal antibody against lysozyme. Expression of group II phospholipase A2 gene was shown using Northern hybridization and in situ hybridization. RESULTS: Lysozyme was present in the secretory granules of most acini, whereas group II phospholipase A2 was seen in a minority of acinar cells, primarily in the central parts of lobules in the main and accessory lacrimal glands. Synthesis of group II phospholipase A2 in the glandular cells was confirmed by Northern hybridization and by in situ hybridization. CONCLUSIONS: There are two specialized cell types in the main and accessory lacrimal glands, one synthesizing group II phospholipase A2 and the other synthesizing lysozyme. These enzymes are important nonspecific antibacterial factors in tears.

Pathogenetic differences between coxsackie A and B virus infections in newborn mice.

Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.

Pre-eclampsia does not change the adhesion molecule status in the placental bed.

During normal placentation trophoblast cells invade maternal tissues and remodel the uterine arteries into low-resistance channels. In pre-eclampsia, trophoblast invasion is impaired and this, along with endothelial dysfunction, has been suggested to play a role in the pathogenesis of pre-eclampsia. We studied the expression of adhesion molecules important for leukocyte extravasation in the placental bed with immunohistochemistry and compared the expression in pre-eclampsia to that in normal pregnancy. Our major finding was that only invasive trophoblasts expressed cutaneous lymphocyte antigen-1 (CLA-1) in the third trimester of pregnancy, whereas villous trophoblasts did not. In the first trimester both villous trophoblasts and invasive trophoblast cells in decidua remained negative for CLA-1. Pre-eclampsia did not change the expression of leukocyte-endothelium adhesion or lymphocyte homing-associated antigens, ICAM-1, ICAM-2, VCAM, P-selectin, E-selectin, L-selectin, CLA-1, CD73, VAP-1 and alphaEbeta7 in the placental bed. Furthermore, pre-eclampsia was not associated with an aberrant accumulation of lymphocytes carrying antigens of any particular known organ-specific homing systems. The results on the unchanged pattern of adhesion molecule expression in pre-eclampsia suggests that there is no major change in the adhesive properties of the endothelium of the placental bed in pre-eclampsia.

Pulmonary alveolar proteinosis and glomerulonephritis in lysinuric protein intolerance: case reports and autopsy findings of four pediatric patients.

Lysinuric protein intolerance is an autosomal recessive disease caused by defective transport of cationic amino acids. Of the 38 lysinuric protein intolerance patients diagnosed in Finland since 1965, four pediatric patients have died. We describe the clinical courses and autopsy findings for these patients. All patients developed acute respiratory insufficiency. In addition to pulmonary hemorrhages, three of the patients had pulmonary alveolar proteinosis and one had cholesterol granulomas. Three patients had a clinically obvious renal insufficiency, but all four showed histologic signs of immune complex-mediated glomerulonephritis. The patients also developed hepatic insufficiency with fatty degeneration or cirrhosis. All patients showed anemia, thrombocytopenia, and a severe bleeding tendency. The bone marrow of three patients was hypercellular, but the amount of megakaryocytes was decreased in two cases. Amyloid was present in the lymph nodes and the spleen. Bone specimens showed osteoporosis. We conclude that pediatric patients with lysinuric protein intolerance are predisposed to develop pulmonary alveolar proteinosis and glomerulonephritis. They are also at risk of protein malnutrition in the active growth phase, probably due to higher requirements for total nitrogen and amino acids.

Inclusion body myositis and paramyxoviruses.

Inclusion body myositis (IBM) is a distinct type of muscle disease. The characteristic electron microscopic findings, intranuclear or intracytoplasmic inclusions composed of microtubular filaments, morphologically resemble paramyxovirus nucleocapsids. These findings and the reported immunoreactivity of the inclusions with mumps virus antibodies have suggested that inclusion body myositis is a chronic virus infection. We analyzed skeletal muscle specimens from three patients with characteristic light microscopic features and electron microscopically verified inclusions of IBM by immunocytochemistry using antibodies raised against members of the paramyxovirus group, and by in situ hybridization with a cRNA probe representing the mumps virus nucleocapsid gene. The specificity of the reactions was demonstrated with infected and uninfected cultured cells. No immunocytochemical staining or hybridization signal was observed in biopsy specimens from IBM patients. These findings speak against a paramyxovirus etiology of IBM.

Ossifying fibromyxoid tumour of soft parts: report of four cases including one mediastinal and one infantile.

Four cases of ossifying fibromyxoid tumour of soft parts are described. One of them was in the mediastinum, a hitherto unreported location of this rare neoplasm. Another was removed from the subcutaneous tissue of the head of a two-year-old girl, the youngest patient so far described. A peculiar feature of this tumour was haphazard spindle cell groups showing smooth muscle differentiation. One tumour was remarkably proliferative with 20 mitotic figures per 10 high power fields and 50% of cells positive for Ki-67 antigen. Immunohistochemical analysis revealed that all the tumours were diffusely positive for vimentin, and focally for S-100-protein. In addition to this the infantile tumour expressed focal alpha-smooth muscle actin, desmin and glial fibrillary acidic protein, while the mediastinal tumour expressed only alpha-smooth muscle actin and the highly proliferative one expressed none of these antigens. Background cells, including histiocytes, lymphocytes and mast cells were numerous. DNA cytometry analysis using both static and flow methods showed that the mediastinal tumour contained two cell clones, while the others were diploid. The proliferative fraction of cells (S plus G2 phases) was prominent in the proliferative and infantile tumours.

Mapping of antigenic sites of coxsackievirus B3 by synthetic peptides.

Peptides presenting predicted antigenic sites of CBV3 capsid proteins and peptide sequences from conserved regions of the nonstructural proteins were synthesized, and rabbit antipeptide sera were tested for their immunoreactivity. Peptides derived from different capsid regions were able to induce production of neutralizing antibodies in rabbits. As measured by EIA, all peptides representing four different proposed antigenic sites were immunogenic, inducing an antibody response against the homologous peptide and purified CBV3 as measured by EIA. Immunization with inactivated CBV3 induced a secondary response especially in rabbits primed with peptides representing polypeptide VP2. Antisera against the nonstructural protein sequences were highly cross-reactive with other enteroviruses, while the capsid peptide antisera were mainly type-specific when tested by immunoblotting against a panel of enteroviruses. Four of the capsid region peptides also exhibited distinct T-cell reactivity in a mouse T-cell proliferation assay.

Immunohistochemical localization of transferrin receptors in junctional and sulcular epithelium of human gingiva.

Transferrin receptors were found on the apical cells of the junctional epithelium and on the basal and adjacent suprabasal cells of the sulcular epithelium, consistent with the rapidly renewing character of these tissues. High density of receptors was found in the coronal portion of the junctional epithelium and on the sulcular cells adjacent to the tooth surface. This may indicate the proliferative potential of such cells rather than their actual division. An inert substratum, stimulating the tooth surface in vitro caused positive expression of the transferrin receptor, but not so extensively as in vivo. Thus factors not present in culture may normally activate the distinct non-differentiating junctional and sulcular cells to express the receptor.

Transformation-specific matrix metalloproteinases (MMP)-7 and MMP-13 are expressed by tumour cells in epidermolysis bullosa-associated squamous cell carcinomas.

BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) have an increased risk of developing rapidly progressive and metastatic cutaneous squamous cell carcinomas (SCC). It is unclear why these SCC behave more aggressively than sporadic SCC. Matrix metalloproteinases (MMP) are a family of endopeptidases that contribute to growth, invasion and metastasis of SCC. The role of MMP in RDEB-associated SCC is not known. OBJECTIVES: To investigate the expression of MMP-7, MMP-13 and MMP-9 in RDEB-associated SCC in comparison with sporadic SCC and Bowen's disease. METHODS: Immunohistochemical analysis of 25 RDEB-associated SCC, 61 sporadic SCC and 28 sporadic lesions of Bowen's disease was carried out using monoclonal antibodies for MMP-7, MMP-9, MMP-13 and E-cadherin and syndecan-1. RESULTS: MMP-7 was detected in all RDEB-associated SCC, in tumour cells within the invasive edge, where E-cadherin and syndecan-1 were markedly diminished or absent. MMP-7 expression was also observed in 98% of sporadic SCC and in 68% of Bowen's diseases. MMP-7 staining was significantly stronger in RDEB-associated SCC than in sporadic SCC, and was most abundant in poorly differentiated tumours. MMP-13 was detected in tumour cells in 96% of RDEB-associated SCC and in all sporadic cutaneous SCC. MMP-9 was detected in the inflammatory cells in all SCC examined. CONCLUSIONS: These results identify MMP-7 and MMP-13 as tumour cell-specific markers for SCC progression and as potential therapeutic targets in RDEB-associated SCC. The pattern of immunolabelling suggests that MMP-7 may shed E-cadherin and syndecan-1 from the SCC cell surface.

Gel electrophoretic analysis of nuclear matrix fractions isolated from different human cell lines.

The nuclear matrix is operationally defined as the structure that remains after nuclei are extracted with nonionic detergent and with high salt and are digested with nucleases. Thus the nuclear matrix protein composition is critically dependent on the isolation conditions. We have compared nuclear matrices isolated from human cell lines by two different methods. First, isolated nuclei were extracted as above to obtain a matrix fraction. This method showed a substantial contamination by cytoplasmic intermediate filaments but immunization of mice resulted in antibodies recognizing nuclei and the mitotic spindle apparatus. Second, a nuclear matrix fraction was made by extracting whole cells as above and dissolving the residue in urea and dialysing against an assembly buffer to precipitate intermediate filament proteins (Fey, E. G. and Penman, S., Proc. Natl. Acad. Sci. USA 1988, 85, 121-125). Such fractions showed complex protein patterns in silver-stained two-dimensional gels for four cell lines: HeLa, MCF-7, SW13 and the U333CG/343MG glioma line. While some proteins in the nuclear matrix fraction were common to all cell lines, others appeared cell-line specific. Two-dimensional gels and the immunoresponse in mice again showed contamination of these preparations with cytoplasmic proteins. These results clearly show the difficulties associated with protein chemical analysis of nuclear matrices: the preparations have substantial cytoplasmic contamination, the polypeptide composition is extremely complex and the yield of individual polypeptides is low. Thus, without further experiments one cannot say which proteins are true nuclear matrix components.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody against human sperm acrosome inhibits sperm penetration of zona-free hamster eggs.

Human spermatozoa were exposed to a monoclonal antibody (C11H), which recognizes sperm acrosin. The antibody was presented to the sperm during capacitation and/or insemination, and its effect on penetration was tested using zona-free hamster eggs. An inhibitory effect on penetration was observed when the antibody was present during insemination but not when it was included only in the capacitation medium. As judged by immunofluorescence microscopy, most of the sperm bound to the egg surface were devoid of acrosomal staining. Some of the bound sperm were stained at their equatorial segments. Sperm that had penetrated the ooplasm did not exhibit immunofluorescence.

An acrosomal antigen of human spermatozoa and spermatogenic cells characterized with a monoclonal antibody.

Monoclonal antibodies were raised against acrosomal antigens of human sperm by immunizing BALB/CA mice with purified ejaculated human spermatozoa. An ELISA-assay, employing glutaraldehyde-fixed spermatozoa as antigen, was used to screen the hybridomas producing anti-human sperm antibodies. Two hybridoma cell-lines produced antibodies which bound to the acrosomal region of spermatozoa. Both gave identical results in preliminary tests and therefore only one was chosen for further experiments. This antibody stained the acrosomal region of fixed but not living spermatozoa by indirect immunofluorescence, indicating an intra-acrosomal localization of the antigen. In acetone-fixed frozen sections of human testis this antigen was expressed only in germ cells in the adluminal compartment of seminiferous tubules. The antigen was clearly visible in round spermatids from the beginning of the cap phase of acrosome development and was also present in premature germ cells which were present in ejaculates and which were in the early stages of acrosome development. By immunochemical analysis this antibody recognized a molecule of 50 K MW as well as other components of 24 to 34 K. The pattern of staining for the antigen was similar in the presence or absence of beta-mercaptoethanol in the sample buffer. The species specificity of the antigen was studied by indirect immunofluorescence using acetone-fixed spermatozoa and the antigen was found to be present in mouse, bovine, ram and boar spermatozoa. This antibody may be useful as an acrosomal marker.

A 210 kDa nuclear matrix protein is a functional part of the mitotic spindle; a microinjection study using SPN monoclonal antibodies.

Six monoclonal antibodies identify a 210 kDa polypeptide which shows a cell cycle specific redistribution from the nucleus to the mitotic spindle. In interphase cells this polypeptide was localized in the nucleus and behaved during differential cell extraction as a component of the nuclear matrix. It accumulated in the centrosome region at prophase, in the pole regions of the mitotic spindle at metaphase and in crescents at the poles in anaphase, and reassociated with the nuclei as they reformed in telophase. Due to its staining pattern we call the protein the Spindle Pole-Nucleus (SPN) antigen. The localization of SPN antigen during mitosis was dependent on the integrity of the spindle since treatment of cells with nocodazole resulted in the dispersal of SPN antigen into many small foci which acted as microtubule organizing centres when the drug was removed. The SPN antigen was present in nuclei and mitotic spindles of all human and mammalian cell lines and tissues so far tested. When microinjected into the cytoplasm or nuclei of HeLa cells, one antibody caused a block in mitosis. Total cell number remained constant or decreased slightly after 24 h. At this time, about half the cells were arrested in a prometaphase-like state and revealed aberrant spindles. Many other cells were multinucleate. These results show that the SPN antigen is a protein associated with mitotic spindle microtubules which has to function correctly for the cell to complete mitosis.

Preferential expression of NuMA in the nuclei of proliferating cells.

Nuclear mitotic apparatus protein (NuMA) has an indispensable function in normal mitosis as an organizer of the mitotic spindle. NuMA is a prominent component of interphase cell nuclear matrix but its role during interphase is largely unknown. We examined the presence of NuMA in several human tissues. The majority of cells were positive for NuMA but a few negative cell types were found, including spermatozoa, superficial keratinocytes, neutrophil granulocytes, syncytiotrophoblasts, and some neurons, fibroblasts, and smooth and skeletal muscle cells. We further investigated the presence of NuMA in a cultured estrogen-dependent human breast cancer cell line and observed the disappearance of nuclear NuMA in the quiescent cells. The percentage of NuMA-positive cells diminished from an initial approximately 100 to 60% during 6 days of culture. The presence of NuMA correlated positively with the presence of proliferation marker Ki-67 antigen and negatively with the culture time, confluence, and size of the cell islets. These results show that some nonproliferating, highly differentiated cell types lack NuMA and that cells may lose their NuMA without dramatic effects on the nuclear shape. This suggests that NuMA may be a nonessential component of the interphase nucleus.