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PubMed2004

Identification of genes expressed in primate primordial oocytes.

BACKGROUND: The factors involved in oocyte survival and transition from quiescence to the growing phenotype remain unknown. Herein we report genes that are differentially expressed in the primordial oocyte revealed by DNA arrays. METHODS: Primordial oocytes were captured selectively in rhesus monkey ovary sections using laser capture microdissection. The RNA was extracted and amplified in two rounds by T7-based linear RNA amplification, fluorescence labelled and then hybridized to human cDNA arrays containing 7680 elements. RNA from human placenta served as a reference sample. RESULTS: Ninety-five genes were found to be consistently expressed at a higher level in primordial oocytes. Expression of several of these genes in the oocyte has been reported before, e.g. deleted in azoospermia (DAZ), prohibitin and transglutaminase 2. Oocyte expression of several novel transcripts revealed on array hybridization, such as gene 33, ubiquitin-conjugating enzyme E2A, G1 to S phase transition 1, growth arrest and DNA damage-inducible (GADD), and dendritic cell-derived ubiquitin-like protein (DC-UbP) was confirmed by in situ hybridization. Some array-identified gene products [integrin beta3, alpha-tubulin, regulatory telomere elongation protein (RAP1) and cellular repressor of EIA-stimulated genes (CREG protein)] were detected in human oocytes by immunofluorescence. Bioinformatic analysis of the oocyte-enriched transcripts reveals a functional profile summarized as follows: cell cycle (14%); transporter (13%); signal transduction (10%); cytoskeletal (7%); transcription factor (5%); immune response (5%); apoptosis-related (5%); RNA processing (5%); and the remainder of miscellaneous categories. CONCLUSIONS: These observations may contribute to the elucidation of molecular pathways involved in oocyte survival and maturation.

Animals↗
PubMedDate not supplied

Marker molecules of human endometrial differentiation can be hormonally regulated under in-vitro conditions as in-vivo.

An established cell culture system of isolated human endometrial stromal and epithelial cells has been used to study the effects of oestrogen and progesterone, as well as their antagonists, upon endometrial cells. Normal hormonal regulation in vivo was investigated simultaneously in endometrial tissue samples taken at different phases of the menstrual cycle. Several marker molecules analysed by immunohistochemistry appeared to depend strongly on endocrine regulation and could be traced in culture. Immunohistochemically, basic parameters of cell biology were identified in vitro, e.g. cell proliferation (Ki-67), adhesion molecules (beta3 integrin) and paracrine factors (leukaemia inhibitory factor). The most reliable parameters to assess hormonal influences were oestrogen and progesterone receptor molecules. Immunohistochemical localization could be improved by molecular biological analysis using RT-PCR. In the presence of oestrogen, a significant expression of hormone receptors was also shown by RT-PCR, and withdrawal of oestrogens and addition of gestagen, i.e. medroxyprogesterone acetate, caused receptor downregulation. Addition of the anti-oestrogen ICI 182.780 to cell-culture medium significantly decreased the synthesis of progesterone receptors.

Cell Differentiation↗
PubMed1992

The gene organization of the human beta 7 subunit, the common beta subunit of the leukocyte integrins HML-1 and LPAM-1.

The integrin beta 7 subunit associates with two alternative alpha subunits termed alpha HML-1 and alpha 4 to give the lymphocyte activation and homing receptors HML-1 and LPAM-1. Overlapping genomic clones that encoded the human beta 7 subunit gene were isolated from cosmid and phage lambda libraries. The coding portion of the gene spanned approximately 10 kb and was composed of 14 exons. Exon 1 (123 bp) encoded 5' untranslated sequences; exon 2 (204 bp) encoded the initiation codon, signal peptide, and 50 amino acid residues of the N-terminus of the mature protein; 7 exons (exons 3-9), ranging in size from 90 bp to 242 bp, encoded most of the extracellular domain proximal to the four cysteine-rich repeats; the region corresponding to the beta 3 arginine-glycine-aspartic acid (RGD)-binding domain was divided amongst exons 4 and 5; the four cysteine-rich repeats were encoded by 3 exons (exons 10-12) with intron insertion into the first and third repeats; exon 13 (209 bp) provided a spacer between the cysteine-rich domains and the transmembrane domain; exon 14 (161 bp) encoded the transmembrane domain and exactly half of the cytoplasmic domain; the remainder of the cytoplasmic domain and most of the 3' untranslated region was contained in the largest 313 bp exon 15. Comparison of integrin beta subunit genes revealed that the gene organization of beta 7 was almost identical to that of beta 2, but had diverged from that of beta 3. Amplification of integrin DNAs directly from genomic DNA, using PCR primers based on beta subunit consensus sequences corresponding to the beta 3 RGD-binding domain, yielded partial gene sequences for the beta 3, beta 5, and beta 6 subunits only. Inspection of the amplified sequences revealed that, as for beta 3, the regions in beta 5 and beta 6 corresponding to the beta 3 RGD-binding domain lacked the intron present in beta 7, beta 1, and beta 2, which divides this region in beta 2 into two subdomains that contribute to subunit assembly. This study provides genetic evidence for at least two major branches to the integrin beta subunit evolutionary tree, with beta 7, beta 2, and probably beta 1 in one branch, and the cytoadhesin beta 3 and probably also beta 5 and beta 6 in the other.

Amino Acid Sequence↗
PubMed1996

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

Animals↗
PubMed1998

Opposing effects of low and high molecular weight kininogens on cell adhesion.

High molecular weight kininogen (HK) blocks cell spreading but not cell attachment to surfaces coated with vitronectin and other ligands of beta3 integrins. We sought to learn the structural basis of this phenomenon. Monoclonal antibodies against the histidine-rich D5 domain in the light chain of 2-chain HK abolished the inhibitory effect of 2-chain HK on spreading of MG-63 osteosarcoma cells on vitronectin-coated tissue-culture plastic. The antibodies were effective only if incubated with 2-chain HK in solution and did not abolish the anti-cell-spreading effect of 2-chain HK that was pre-adsorbed to tissue-culture plastic. Exposure of an epitope in the histidine-rich domain was less when HK was adsorbed to tissue-culture plastic (oxidized polystyrene) than when it was adsorbed to ELISA plastic (untreated polystyrene). Loss of the epitope correlated with increased anti-cell-spreading activity of HK on tissue-culture plastic. The light chain of 2-chain HK containing D5 and that containing recombinant D5 both had anti-cell-spreading activity, but only when present in solution during adhesion assays. Pre-adsorption of recombinant D5 to tissue-culture plastic resulted in a surface on which adsorbed 2-chain HK had little anti-cell-spreading activity. Binding study revealed that HKa bound to immobilized vitronectin. The histidine-rich D5 domain of light chain of HK was identified as one of the binding sites of vitronectin, suggesting that the masking of the RGD cell-binding site of immobilized vitronectin is the molecular mechanism of anti-cell-spreading effect of HKa. In contrast, low molecular weight kininogen (LK), which lacks D5, augmented cell spreading on vitronectin-coated tissue-culture plastic. Thus, HK and LK have opposing effects on VN-dependent cell adhesion. The augmenting effect of LK was greater if LK was preincubated with cells or adsorbed to the surface at pH>7.0. Analysis of fragments of LK and antibody inhibition studies localized the cell-adhesion activity to the D3 domain that is common to LK and HK. These findings indicate that the D5 domain mediates the adsorption of HK or 2-chain HK to vitronectin substratum in anti-adhesive conformations, i.e., masking of the RGD cell-binding site of vitronectin. Such conformers inhibit cell spreading on vitronectin even though a cell-adhesion site is present in D3.

Antibodies, Monoclonal↗
PubMed2003

Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor.

Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to promote thrombus formation in high shear environments. We found that thrombospondin-1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin-1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s(-1). Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin-1. Platelet adhesion to thrombospondin-1 is not mediated via beta3-integrins or GPIa. CD36 partially mediates the adhesion of pre-activated platelets. We identified GPIb as high shear adhesion-receptor for thrombospondin-1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin-1-GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.

Humans↗
PubMed2001

Adenoviral vector transfection into the pulmonary epithelium after cecal ligation and puncture in rats.

BACKGROUND: Adenoviral-targeted gene delivery to respiratory epithelium can augment production of specific proteins. Therefore, it may be valuable in treating the acute respiratory distress syndrome. The authors tested the hypothesis that adenoviral vector uptake after cecal ligation and double puncture in rats, an animal model of the acute respiratory distress syndrome, is higher than that observed in controls that did not undergo operation ("nonoperated") or those that underwent a sham operation ("sham-operated"). METHODS: Adenoviruses expressing green fluorescent protein or Lac-Z were delivered into the lungs of anesthetized rats via tracheal catheter. Animals were killed 24 or 48 h later. Histopathology and green fluorescent protein expression were examined using light of fluorescence microscopy. Cellular localization of Lac-Z was determined with electron microscopy or semithin sectioning. Viral receptor density and localization were determined using imunoblotting and immunohistochemistry. RESULTS: After cecal ligation and double puncture, rats were hypoxic and tachypneic. Alveoli were segmentally consolidated, contained proteinaceous debris and neutrophils, and had thickened septa. Administration of adenoviruses to rats that were sham-operated or underwent cecal ligation and double puncture resulted in high levels of marker protein expression in cells lining alveoli. Use of 3 x 10(11) plaque-forming units instead of 3 x 10(12) plaque-forming units resulted in similar levels of green fluorescent protein expression with negligible viral-mediated lymphocytic infiltration. Semithin section and electron microscopy revealed expression primarily localized to type II alveolar cells. Abundance of alpha(v)beta3 integrins and human coxsackie-adenovirus receptor (receptors that modulate viral attachment and internalization) was increased after cecal ligation and double puncture, predominantly in type II pneumocytes. CONCLUSIONS: Cecal ligation and double puncture induces histologic and functional changes consistent with the acute respiratory distress syndrome, increases surface expression of viral receptors, and enhances adenoviral-mediated gene transfer.

Adenoviridae↗
PubMed1997

Antagonism of the GPIIb/IIIa receptor with the nonpeptidic molecule BIBU52: inhibition of platelet aggregation in vitro and antithrombotic efficacy in vivo.

The glycoprotein (GP) IIb/IIIa (the alphallb beta3 integrin) found on platelets binds fibrinogen or von Willebrand factor when the platelet is activated, thereby mediating the aggregation of platelets. Blockade of the GPIIb/IIIa should prevent platelet aggregation independent of the substance or substances responsible for activating the platelets. This comprehensive inhibition of platelet aggregation is thought to be an effective therapeutic approach to various clinical thromboembolic syndromes. This study investigated the platelet inhibition provided by blocking GPIIb/IIIa by using a new nonpeptidic molecule, BIBU52, in both in vitro and in vivo models. BIBU52 competes with [125I]fibrinogen for binding sites on human platelets in a Ca2+ and pH-dependent manner with a 50% inhibitory concentration (IC50) of 35 +/- 12 nM. BIBU52 inhibited the aggregation of human platelets in platelet-rich plasma induced by collagen (1-2 microg/ml), adenosine diphosphate (ADP; 2.5 microM), and a thrombin receptor-activating peptide (TRAP; SFLLRNPNDKYEPF-NH2; 25 microM) with IC50 values of 82, 83, and 200 nM, respectively. The inhibition of platelet aggregation by BIBU52 was found to be highly species dependent. BIBU52 inhibited aggregation in plasma from rhesus and marmoset monkeys with an IC50 of 150 nM but was totally ineffective in rat plasma. The selectivity of BIBU52 for inhibiting GPIIb/IIIa in comparison with other adhesion molecules was investigated in a human endothelial cell adhesion assay. The adhesion of human endothelial cells to matrices of vitronectin, fibronectin, collagen I, or laminin was not affected by concentrations as high as 100 microM BIBU52; thus BIBU52 demonstrates a high selectivity for the human GPIIb/IIIa. The antithrombotic effect of BIBU52 in vivo was investigated in three animal models of recurrent arterial thrombus formation. In the guinea pig aorta, BIBU52 inhibited thrombus formation dose dependently, with lack of thrombus formation for 1 h after a bolus dose of 1.0 mg/kg i.v.. Both acetylsalicylic acid and dazoxiben were less effective in this model. In pigs with recurrent thrombus formation in the carotid artery, 1.0 mg/kg i.v. also inhibited thrombus formation. Heparin was not effective in the pig, and acetylsalicylic acid was only partially effective. In the pig, the dose of 1.0 mg/kg i.v. BIBU52 also was associated with a 70% inhibition of collagen-induced platelet aggregation ex vivo but with only a transient prolongation of sublingual bleeding time to a maximum of 2.5-fold and without other hemodynamic effects. In the marmoset monkey, a dose of 10 microg/kg i.v. could abolish recurrent arterial thrombosis. Hemodynamic effects of BIBU52 in anesthetized pigs were not detected in doses < or = 10 mg/kg. These data demonstrate that BIBU52 is a potent and selective antagonist of the human GPIIb/IIIa receptor and capable of substantial inhibition of platelet aggregation in vitro and ex vivo as well as inhibition of arterial thrombus formation in vivo in animal models of thrombosis.

Animals↗
PubMed2005

Snail-regulated genes in malignant melanoma.

The demonstration that zinc-finger transcriptional repressors can control E-cadherin expression in epithelial cells has provided a new avenue of research in the field of epithelial-mesenchymal transition (EMT). One of these zinc-finger molecules is the transcription factor Snail, which controls gastrulation and neural crest EMT in different species. Additionally, Snail is involved in the development of malignant melanoma where a dramatic change in E-cadherin expression is an important early step for melanoma progression. For this study, a human cancer cDNA array was used which includes genes involved in cancer development and progression. Using the array we compared the gene expression pattern of the melanoma cell line Mel Im with a Mel Im cell clone stable transfected with antisense (as) SNAIL cDNA. We validated the significant differences of the expression of genes on mRNA level. Primarily, we observed changes in the expression of genes involved in EMT. Quantitative real-time polymerase chain reaction showed a down-regulation of MMP-2, EMMPRIN, SPARC, TIMP-1, t-PA, RhoA and Notch4 expression and a re-induction of E-cadherin expression in the as Snail cell clones. Furthermore, we measured the expression of integrin beta3, NM23b and RhoB. Additionally, we investigated whether the selected genes are influenced only through Snail or if E-cadherin can influence the expression of these genes. In summary, all examined genes which are influenced through Snail have a regulatory function in EMT processes as does Snail itself. The Snail target gene E-cadherin has no regulatory function with respect to the selected genes.

Cadherins↗
PubMed2006

Expression pattern of osteopontin in endometrial carcinoma: correlation with expression of the adhesion molecule CEACAM1.

Osteopontin (OPN) and CEACAM1 have diverse biological functions in the uterus and placenta throughout the estrous cycle and pregnancy and have been shown to interact with integrin beta3. OPN is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. Recently we showed the expression pattern of OPN in gestational trophoblastic tumors. CEACAM1 is an adhesion molecule of the carcinoembryonic antigen family that we have recently found to be expressed in endometrial cancer and that has been shown to be down-regulated in colorectal, prostate, and breast cancer. In this study, immunohistochemistry and immunofluorescence with specific antibodies were performed on a series of 20 normal endometrial samples, 17 endometrial hyperplasias, and 43 endometrial carcinomas (28 endometrioid, 10 serous, and 5 clear cell carcinomas) to investigate the expression pattern and cell-type specific localization of OPN and to correlate it with the expression of CEACAM1. In addition, Western blot was performed on normal human endometrium and endometrial neoplasia. Strong OPN expression with a consistent cytoplasmic localization in epithelial glandular cells was observed in the normal human endometrium in 80% of the samples of the proliferative and secretory phase (score 8-12). Similar results could be found in endometrial hyperplasias. Strong expression of OPN could be observed in 29 (67.4%) of the 43 analyzed endometrial carcinomas. Of the 43 analyzed tumors, 18 (41.8%) were in the high score (8-12) category with a strong OPN expression level; 11 of 43 (25.5%) showed a moderate score (4-7) category. In endometrioid carcinoma with increasing malignancy grade, increasing areas with low OPN expression level or complete loss of OPN expression could be observed. In contrast, serous tumors showed a strong OPN expression level. Similar results could be found in Western blot analysis. CEACAM1 showed similar results and could be found to be coexpressed with OPN in normal human endometrium and in endometrial neoplasia as we showed using immunofluorescence. In this study, the different expression patterns of OPN in endometrial tumors could additionally support the biological diversity of endometrioid and serous carcinomas together with other markers. We suggest that OPN might play a different role in the pathogenesis of endometrial cancer (possibly as a functional complex with CEACAM1) and could be relevant for invasive growth of such lesions.

Adenocarcinoma, Scirrhous↗
PubMed2006

Platelet-activating factor synthesis and response on pancreatic islet endothelial cells: relevance for islet transplantation.

BACKGROUND: Recent data suggest that donor intraislet endothelial cells may survive islet transplantation and participate to the events that influence islet engraftment. However, the mechanisms that regulate islet endothelial behavior in this setting are poorly known. METHODS: We obtained immortalized human (hIECs) and mouse (mIECs) islet endothelial cells by transfection with SV40-T-large antigen and studied the synthesis and response to Platelet-activating factor (PAF), a multipotent phospholipid that acts as endothelial mediator of both inflammation and angiogenesis. RESULTS: HIECs showed typical endothelial markers such as expression of vWF, CD31, and CD105, uptake of acetylated-LDL and binding to ULE-A lectin. Moreover, they expressed nestin, the PAF-receptor and possess surface fenestrations and in vitro angiogenic ability of forming tubular structures on Matrigel. Likewise, mIECs showed expression of vWF, CD31, nestin, PAF-receptor and CD105, and uptake of acetylated-LDL. HIECs and mIECs rapidly produced PAF under stimulation with thrombin in a dose-dependent way. Exogenous PAF or thrombin-induced PAF synthesis increased leukocyte adhesion to hIECS and mIECs and cell motility of both endothelial cell lines. Moreover, PAF or thrombin-induced PAF synthesis accelerated in vitro formation of vessel-like tubular structures when hIECs are seeded on Matrigel. Notably, gene-microarray analysis detected up-regulation of beta3 integrin gene on hIECs stimulated with PAF, that was confirmed at the protein level. CONCLUSIONS: Based on the novel development of immortalized islet endothelium, these results suggest that PAF may have a dual role that links inflammation to angiogenesis in the early events of islet transplantation.

Animals↗
PubMed1998

Induction of neutralizing antibodies by synthetic peptides representing the C terminus of coxsackievirus A9 capsid protein VP1.

The arginine-glycine-aspartic acid motif at the C terminus of coxsackievirus A9 capsid protein VP1 has been shown to play a role in specific attachment of the virus to alpha(v)beta3 integrin on the host cell surface. The C-terminal region of the VP1 protein has also been shown to be highly antigenic by using peptide scanning techniques. To find out whether this region contains a neutralizing epitope, three overlapping peptides covering the C-terminal end of VP1 were synthesized and rabbit antisera were raised against these peptides. Neutralization of the virus was observed with all three antipeptide antisera in A549 cells and with two antisera in RD cells.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular imaging and therapy directed at the neovasculature in pathologies. How imaging can be incorporated into vascular-targeted delivery systems to generate active therapeutic agents.

We have discussed the impact of molecular imaging on clinical and preclinical medicine. We have presented the potential problems of delivering the effective therapeutic dose and the properties that can help contribute to the drug efficacy. The rationale for the design of new antiangiogenic agents that can be used for imaging and therapy was presented. Finally, results from imaging and targeted nanoparticle based therapies were presented. In vivo imaging of angiogenic tumors using anti-alpha(v)beta3 -targeted polymerized vesicles composed of the murine antibody LM609 attached to NPs labeled with the MR contrast agent gadolinium in the V2 carcinoma model in rabbits. MRI studies using this targeted contrast agent revealed large areas of alpha(v)beta3 integrin expression in tumor-associated vasculature that conventional MRIs failed to show. Other investigators have used microemulsions conjugated to an antibody targeted against alpha(v)beta as imaging agents. These materials also show contrast enhancement of tumor vasculature undergoing angiogenesis. Other markers, such as the PECAM-1 (CD-31), VCAM-1 (CD54) and VEGF receptor (flk-1), have been shown to be upregulated on tumor endothelium and associated with angiogenesis but have not been used in imaging studies. Furthermore, by modification of the NPs, we were able to use this imaging agent as an antiangiogenic gene delivery system. The results from these studies are very promising and are being further pursued.

Angiogenesis Inhibitors↗
PubMed2004

The C terminus of the nuclear protein NuMA: phylogenetic distribution and structure.

The C terminus of the nuclear protein NuMA, NuMA-CT, has a well-known function in mitosis via its proximal segment, but it seems also involved in the control of differentiation. To further investigate the structure and function of NuMA, we exploited established computational techniques and tools to collate and characterize proteins with regions similar to the distal portion of NuMA-CT (NuMA-CTDP). The phylogenetic distribution of NuMA-CTDP was examined by PSI-BLAST- and TBLASTN-based analysis of genome and protein sequence databases. Proteins and open reading frames with a NuMA-CTDP-like region were found in a diverse set of vertebrate species including mammals, birds, amphibia, and early teleost fish. The potential structure of NuMA-CTDP was investigated by searching a database of protein sequences of known three-dimensional structure with a hidden Markov model (HMM) estimated using representative (human, frog, chicken, and pufferfish) sequences. The two highest scoring sequences that aligned to the HMM were the extracellular domains of beta3-integrin and Her2, suggesting that NuMA-CTDP may have a primarily beta fold structure. These data indicate that NuMA-CTDP may represent an important functional sequence conserved in vertebrates, where it may act as a receptor to coordinate cellular events.

Amino Acid Sequence↗
PubMed2004

Integrin expression in cells of the intervertebral disc.

In this study, we investigated the profile of integrin expression in human and porcine intervertebral disc tissue. Differences in extracellular matrix composition between anulus fibrosus (AF) and nucleus pulposus (NP) regions of the disc, as well as differences in cellular responses to environmental stimuli, suggest a role for integrins in presenting matrix signals that may mediate these responses. Human disc tissue and porcine AF and NP tissue were stained with antibodies to alpha integrin subunits 1-6, V and IIb, and beta integrin subunits 1-6 and graded for evidence of positive staining on a scale from 0 (no staining) to 3 (high incidence of staining). Human tissue expressed alpha and beta integrin subunits shown to be present in articular cartilage, including alpha(1), alpha(5) and alpha(V). Porcine AF tissue expressed similar integrin subunits to human disc, with both expressing alpha(1), alpha(5), beta(1), beta(3) and beta(5) subunits, whereas porcine NP tissue expressed higher levels of alpha(6), beta(1) and beta(4) than AF tissue. The expressed subunits are known to interact with proteins including collagens, fibronectin and laminin; however, additional studies will be required to characterize the interactions of the integrin subunits with specific matrix constituents, as well as their specific involvement in regulating environmental stimuli.

Animals↗
PubMed1994

Two human melanoma cell-line variants with enhanced in vivo tumor growth and metastatic capacity do not express the beta 3 integrin subunit.

The alpha v beta 3 integrin complex is thought to play an important role in in vivo melanoma tumor growth and metastasis. However, not all human metastatic melanomas, present in lymph node biopsies, express alpha v beta 3. In this study, we have investigated the possibility that certain melanoma cell lines can grow aggressively in vivo in the absence of alpha v beta 3 expression. Established human melanoma cell lines (M3Da., M4Beu.) were isolated from an achromic skin metastasis or lymph nodes. Two stable variants (7GP, T1P26), derived from a poorly metastatic M4Beu. melanoma cell line, were isolated by sequential selection for spontaneous metastasis formation in an immunosuppressed newborn rat model. Flow-cytometry analysis shows an absence of the beta 3 integrin subunit (less than 2% of parental levels) in the two variant melanoma cell lines (7GP, T1P26) compared to M3Da. and M4Beu. cell lines which express a relatively high number of beta 3 subunits. The expression levels of the integrin subunits beta 1, beta 5, beta 6 and alpha v were found to be similar for all four melanoma cell lines. Northern blot analysis confirmed the absence of beta 3 in 7GP or T1P26 cell lines and its presence in M3Da. and M4Beu. Moreover, similar levels of alpha v transcript were present in the four melanoma cell lines. The functional effect of the absence of beta 3 was investigated by subcutaneously implanting the variants and the melanoma cell lines in nude mice. Variant 7GP and T1P26 cell lines yielded tumors which were larger and grew at a faster rate than tumors in M3Da. or M4Beu. cell lines. The beta 3 integrin subunit was not detectable on the surface of cells harvested from tumors after 20 or 35 days. Similarly, subcutaneous innoculation of the two variants into immunosuppressed newborn rats gave rise to extensive spontaneous lung metastases compared to the M4Beu. cell line. These results provide evidence that a population of melanoma cells can grow aggressively in vivo and metastasize in the absence of beta 3 or alpha v beta 3 integrin complex. Our results may have clinical relevance and suggest that certain types of melanomas in patients may grow and spread in the absence of the alpha v beta 3 integrin complex.

Animals↗
PubMedDate not supplied

Localization of adhesion molecules on human spermatozoa by fluorescence microscopy.

The expression of adhesion molecules on human spermatozoa of healthy probands was analysed. The localization patterns of adhesion molecules (AM) on the spermatozoal surface were documented by fluorescence microscopy. Spermatozoa were incubated with antibodies against alpha 1 (CD49a), alpha 2 (CD49b), alpha 3 (CD49c), alpha 4 (CD49d), alpha 5 (CD49e), alpha 6 (CD49f) chains of beta 1 integrins, beta 1 (CD29), beta 2 (CD18), alpha V (CD51), beta 3 (CD61) and beta 4 integrin chains, the LFA-3 (Lymphocyte function antigen, CD58) from the immunoglobulin superfamily and the extra-cellular matrix proteins laminin, fibronectin and collagen IV. For collagen IV, alpha 1 and alpha 2 chains no expression could be noticed. Laminin was detected at the acrosomal membrane, fibronectin and beta 4 chain mainly at the equatorial membrane. The fibronectin receptors alpha 3, alpha 4 and alpha 5 chains of the beta 1 integrins were mainly located on acrosomal and equatorial membrane areas. Laminin receptor alpha 6 chain was located postacrosomal and less frequently acrosomal. beta 2 chain and vitronectin receptors alpha V and beta 3 chains had a mainly postacrosomal localization pattern. LFA-3 was found constantly on postacrosomal membrane areas. Double staining technique was used to prove the simultaneous occurrence of fibronectin and its integrin receptors alpha 3, alpha 4 and alpha 5 chains and of alpha V and beta 2 chains on spermatozoa. The localization patterns of integrins on double stained spermatozoa were similar to the patterns described for single stained spermatozoa. The localization of fibronectin appeared to be influenced by the presence of integrins: the typical equatorial fibronectin band disappeared in case of an equatorial localization of integrins.

Antigens, CD↗
PubMed1995

Integrin distribution in gastric carcinoma: association of beta 3 and beta 5 integrins with tumor invasiveness.

Immunolocalization of a variety of integrins using monoclonal antibodies against beta 3, beta 5, alpha 3 beta 1 and alpha 6, and polyclonal antibodies against vitronectin receptor (alpha v beta 3) and beta 1 were investigated on PLP-fixed paraffin sections of 19 cases of advanced gastric carcinomas. The beta 5 integrin, which pairs only with the alpha v subunit, was positive in seven cases (37%), and was associated closely with scirrhous invasion (P < 0.05). beta 5 was positive chiefly in the cytoplasm of carcinoma cells and infrequently in cell membranes. beta 3, which is another subunit pairing with alpha v, was positive in six cases (32%), and tended to be associated with scirrhous invasion (P < 0.01). beta 3 was also located chiefly in the cytoplasm. Five of the seven beta 5-positive cases showed coexpression of beta 3. Polyclonal antibodies to alpha v beta 3 also showed a significant difference among the amounts of stroma (P < 0.05). Anti-beta 1 antibodies showed clear positivity in many cases (89%). Of the beta 1 integrins, alpha 3 beta 1 was positive in a few cases (26%) without any preferential pattern, and laminin receptor subunit alpha 6 stained on cell membranes of neoplastic epithelia in many cases of carcinoma (89%) except for mucinous carcinoma These distinctive patterns of integrin positivity indicate a close association of beta 5 and beta 3 expression with scirrhous invasion in gastric carcinoma.

Adult↗