Biomedical subjects

C Van Waes

Publications and source records attributed to C Van Waes.

At least 37 records · Page 2Linked to original sources

PubMed1999

The host environment promotes the constitutive activation of nuclear factor-kappaB and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma.

We reported previously that tumor cells isolated from metastases of the in vitro transformed squamous cell carcinoma line Pam 212 exhibit an elevation in constitutive production of proinflmmatory cytokines interleukin (IL)-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and KC (the murine homologue of chemokine Gro-alpha). The basis for constitutive expression of these cytokines after tumor progression in vivo is unknown. Regulation of the expression of these proinflammatory cytokines involves transcription factor nuclear factor kappaB (NF-kappaB), which can be activated by cytokines such as tumor necrosis factor (TNF)-alpha. In this study, we compared the constitutive and TNF-alpha-induced expression of proinflammatory cytokines in parental Pam 212 and metastatic LY-2 and LY-8 cell lines and determined the relationship of cytokine expression to activation of NF-kappaB. We found that the metastatic cell lines exhibited an increase in constitutive and TNF-alpha-inducible expression of proinflammatory cytokines when compared with parental Pam 212 cells. The increased cytokine expression was associated with an increase in constitutive and TNF-alpha-inducible activation of NF-kappaB as demonstrated by electrophoretic mobility shift assay and luciferase-reporter gene assay. Constitutive nuclear localization of NF-kappaB p65 was observed in LY-2 and LY-8 cells in culture and in tumor specimens but rarely in Pam 212 cells, consistent with the constitutive activation of NF-kappaB in tumor cels after selection in vivo. Induction of NF-kappaB by TNF-alpha was inhibited by the addition of protease inhibitors calpain inhibitor I and N-tosyl-phechloromethyl ketone and antioxidant 1-pyrrolidinecarbodithioic acid, whereas constitutive activation of NF-kappaB and cytokine KC mRNA expression was inhibited by N-tosyl-phechloromethyl ketone alone. Overexpression of a human Ikappa(B)alpha dominant suppresser in Pam 212 cells inhibited TNF-alpha-induced NF-kappaB binding activity and KC expression. These data indicate that activation of NF-kappaB contributes to increased expression of proinflammatory cytokines during metastatic tumor progression of squamous cell carcinoma, and that distinct mechanisms may be involved in the regulation of constitutive and TNF-alpha-induced activation of NF-kappaB in squamous cell carcinoma.

Animals↗

PubMed1999

Constitutive activation of transcription factors NF-(kappa)B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines.

We previously reported that human head and neck squamous cell carcinomas (HNSCCs) express the pro-inflammatory and pro-angiogenic cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in vitro and in vivo. The promoter region of the genes encoding these cytokines include binding sites for the transcription factors nuclear factor (NF) kappaB/Rel A, activator protein-1 (AP-1), and CCAAT enhancer-binding protein beta (C/EBPbeta, or NF-IL6), which have been reported to contribute to activation of these cytokine genes. In the study presented here, we examined the activation, composition, and function of these transcription factors in HNSCC cell lines that express pro-inflammatory cytokines, by using electrophoretic mobility shift and reporter-gene assays. Constitutive activation of NF-kappaB, AP-1, and NF-IL6 DNA-binding proteins was detected. Supershift analysis with antibodies specific for NF-kappaB, AP-1, and NF-IL6 binding proteins showed that the NF-kappaB-binding protein included p65/Rel A and p50; AP-1 activity included c-jun, junB, junD, and Fra-1; and NF-IL6 included C/EBPbeta. Mutational analysis of the NF-kappaB, AP-1, and NF-IL6 sites in the IL-8 promoter region showed that NF-kappaB and AP-1 sites contributed to constitutive IL-8 reporter activity in HNSCC. HNSCC lines that exhibited increased IL-8 secretion relative to simian virus 40-immortalized and primary keratinocyte cell lines also demonstrated a concordant increase in NF-kappaB reporter activity relative to nonmalignant keratinocytes. We concluded that the early transcription factors NF-kappaB, AP-1, and NF-IL6 are constitutively activated in human HNSCC cell lines and that NF-kappaB and AP-1 promote expression of the pro-inflammatory and pro-angiogenic cytokine IL-8 in HNSCC. The demonstration of the activation of these transcription factors will be helpful in defining the identity and role of these and other early gene products that contribute to pathogenesis of the malignant phenotype in HNSCC and in defining potential targets for pharmacologic and molecular therapy of HNSCC. Mol. Carcinog. 26:119-129, 1999. Published 1999 Wiley-Liss, Inc.

CCAAT-Enhancer-Binding Proteins↗

PubMed1999

Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer.

Altered immune, inflammatory, and angiogenesis responses are observed in patients with head and neck squamous cell carcinoma (HNSCC), and many of these responses have been linked with aggressive malignant behavior and a decrease in prognosis. In this study, we examined the hypothesis that HNSCC cells produce cytokines that regulate immune, inflammatory, and angiogenesis responses. We identified important regulatory cytokines in supernatants of well-defined and freshly cultured HNSCC cell lines by ELISA and determined whether these cytokines are detected in tumor cell lines and tissue specimens by immunohistochemistry. The serum concentration of the cytokines and cytokine-dependent acute phase inflammatory responses (i.e., fibrinogen, C-reactive protein, and erythrocyte sedimentation rate) from patients with HNSCC was determined, and the potential relationship of serum cytokine levels to tumor volume was analyzed. Cytokines interleukin (IL)-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor were detected in similar concentration ranges in the supernatants of a panel of established University of Michigan squamous cell carcinoma (UM-SCC) cell lines and supernatants of freshly isolated primary HNSCC cultures. Evidence for the expression of IL-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and VEGF in HNSCC cells within tumor specimens in situ was obtained by immunohistochemistry. In a prospective comparison of the cytokine level and cytokine-inducible acute-phase proteins in serum, we report that cytokines IL-6, IL-8, and VEGF were detected at higher concentrations in the serum of patients with HNSCC compared with patients with laryngeal papilloma or age-matched control subjects (at P < 0.05). The serum concentrations of IL-8 and VEGF were found to be weakly correlated with large primary tumor volume (R2 = 0.2 and 0.4, respectively). Elevated IL-1- and IL-6-inducible acute-phase responses were also detected in cancer patients but not in patients with papilloma or control subjects (at P < 0.05). We therefore conclude that cytokines important in proinflammatory and proangiogenic responses are detectable in cell lines, tissue specimens, and serum from patients with HNSCC. These cytokines may increase the pathogenicity of HNSCC and prove useful as biomarkers or targets for therapy.

Acute-Phase Reaction↗

PubMed1998

Effects of interleukin-1alpha, interleukin-1 receptor antagonist, and neutralizing antibody on proinflammatory cytokine expression by human squamous cell carcinoma lines.

Proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte macrophage colony-stimulating factor (GM-CSF) have been detected in tumor specimens and primary cell cultures from patients with head and neck squamous cell carcinoma. IL-1alpha has been reported to play an important role in inducing the expression of cytokines IL-6, IL-8, and GM-CSF during inflammation. We examined whether these cytokines are expressed together in five primary and seven established UM-SCC cell lines, and we also examined the effects of IL-1alpha, IL-1 receptor antagonist or neutralizing antibody (Ab) upon expression of this repertoire of proinflammatory cytokines in established UM-SCC lines. Secretion of proinflammatory cytokines IL-1alpha, IL-6, IL-8, and GM-CSF was detected by ELISA in both the primary and established UM-SCC lines. Constitutive expression of specific mRNAs for these cytokines was confirmed in the UM-SCC lines by reverse transcriptase-PCR and Northern blot analysis. Addition of recombinant IL (rIL)-1alpha but not rIL-6 induced a dose-dependent increase in IL-8 and GM-CSF production. IL-1 receptor antagonist (IL-RA) or anti-IL-1 neutralizing Ab could completely inhibit the rIL-1alpha-inducible increase in IL-8 and GM-CSF expression, but the inhibitors had a negligible effect on the constitutive level of production of the cytokines. Transfer and expression of the IL-1alpha gene in a low-cytokine-producing cell line, UM-SCC-38, induced IL-8 and GM-CSF expression, but this expression was also not inhibited by IL-1RA or anti-IL-1 neutralizing Ab. We conclude that IL-1alpha can enhance the expression of cytokines IL-8 and GM-CSF in UM-SCC cell lines but that constitutive expression of these cytokines by UM-SCC is not inhibited by exogenous IL-1RA or neutralizing Ab.

Adult↗

PubMed1998

Differential expression of the murine Ly-6A/E antigen homolog of human squamous cell carcinoma antigen E48 during malignant transformation and tumor progression of squamous cell carcinoma line Pam 212.

The mRNA differential display method detected increased expression of the mRNA of an immune recognition antigen known as Ly-6A/E after malignant transformation of the murine squamous cell carcinoma line Pam 212. Subsequent loss of expression of Ly-6A/E occurred with metastatic tumor progression of Pam in vivo. Ly-6 molecules have been implicated in immune cell recognition and signal transduction and are homologous to the human E48 SCC antigen that has been shown to be involved in cell-cell recognition. These observations suggest that loss of Ly-6A/E antigen may contribute to tumor progression and metastasis of squamous cell carcinoma through decreased tumor-lymphocyte or tumor-tumor recognition.

Animals↗

PubMed1998

The host environment promotes the development of primary and metastatic squamous cell carcinomas that constitutively express proinflammatory cytokines IL-1alpha, IL-6, GM-CSF, and KC.

Human and murine squamous cell carcinomas (SCC) have been reported to produce proinflammatory cytokines IL-1alpha, IL-6, GM-CSF, and IL-8 or KC. Production of individual members of the proinflammatory cytokine family has been associated with increased tumor growth or metastasis in a variety of neoplasms. In this study, we determined whether the expression of these cytokines occurs as a result of the events of cellular transformation or culture, or is promoted by interaction of neoplastic cells with factors or cells in the host environment. We compared the expression of proinflammatory cytokines following the spontaneous transformation of murine keratinocytes in vitro, and following the formation of tumors and metastases from these transformed keratinocytes in syngeneic recipients in vivo. Using sensitive ELISA assays, we found that cultures of the in vitro transformed Balb/c SCC line Pam 212 do not produce elevated levels of proinflammatory cytokines IL-1alpha, IL-6, GM-CSF and KC, indicating that transformation or culture alone is insufficient to account for the level of cytokine expression detected in patient and experimental tumors. In contrast, Pam reisolates from primary and metastatic tumors were obtained which constitutively produce markedly elevated levels of cytokines IL-1alpha, IL-6, KC and GM-CSF. The increase in the expression of these cytokines by SCC in vivo occurred independent of T and B lymphocyte-mediated immunity, since increases in expression of the cytokines was observed in lines reisolated from immunodeficient athymic nude and SCID Balb/c congenic mice. The increased expression of cytokines appeared to result from additional events in vivo, rather than due to selection of a pre-existing cytokine-producing subpopulation, since clones of the parental cell line expressed lower cytokine levels than cloned reisolates, and clones of the non-secreting parental cell line that formed tumors in vivo secreted elevated levels of cytokines following reisolation. We conclude that the development of SCC that express proinflammatory cytokines is promoted by tumor-host interaction(s) that are independent of specific T and B cell immunity.

Animals↗

PubMed1998

A human vascular endothelial cell model to study angiogenesis and tumorigenesis.

Endothelial cell biology has recently been the subject of considerable interest in thrombosis and cancer research. However, the successful establishment of immortalized human endothelial cells which retain differentiated cell characteristics has been rare. We have successfully established immortalized human umbilical vein endothelial cells (HUVECs) by human papilloma virus (HPV)-16 E6-E7. HPV-16 E6, E7 and E6-E7 were successfully introduced into HUVEC cells. Both E6 and E7 cultures had an extended lifespan but eventually underwent senescence. E6-E7 cultures 4-5-2G, however, acquired an indefinite lifespan in culture but did not undergo malignant conversion. Telomerase activity was not detected in either E6 or E7 cultures; however, telomerase was detected in E6-E7 4-5-2G cells. The cells exhibited a 'cobblestone' morphology and developed a capillary-like tube structure upon reaching confluence. The 4-5-2G line expressed Factor VIII related antigen and took up DiI-Ac-LDL as markers of endothelial origin. The line expressed integrin subunits (alpha(v)beta3, alph(v)beta5, beta1, alpha2, alpha3, beta4 and alpha6) consistent with an endothelial origin. The higher passage of 4-5-2G line showed a similar intensity of integrin immunostaining to that of primary HUVECS. Subsequent infection of these immortal cells with the Kirsten murine sarcoma virus which contains an activated K-ras oncogene induced morphological transformation that led to the acquisition of invasion capability and neoplastic properties. Telomerase was also detected in the tumorigenic v-Ki-ras transformed cell line. These cell lines should be useful for studies of the molecular mechanisms underlying normal and neoplastic endothelial cell proliferation and migration, and might also provide an in vitro model for development of pharmacologic and gene therapy for cardiovascular thrombosis and cancer.

Cell Line, Transformed↗

PubMed1998

Intravenous delivery of 5'-iododeoxyuridine during hyperfractionated radiotherapy for locally advanced head and neck cancers: results of a pilot study.

OBJECTIVES: Locally advanced cancers of the head and neck require aggressive treatment, often with limited effectiveness and significant toxicity and morbidity. This pilot study was designed to assess tolerance using combined hyperfractionated radiotherapy and the halogenated pyrimidine radiosensitizer 5'-iododeoxyuridine (IdUrd). STUDY DESIGN: This was a prospective single-arm study open to patients with advanced head and neck cancers that had a poor chance of control with conventional radiation therapy. Patients were treated with hyperfractionated radiation therapy at standard doses in combination with an IdUrd infusion and observed for tumor response and normal tissue tolerances. METHODS: Radiation therapy was delivered in fractions of 1.2 Gy or 1.5 Gy twice daily to a total dose in the range of 70 to 76 Gy. IdUrd was delivered as an intravenous infusion (1000 mg/m2 per day) for a maximum of 14 days at the beginning and then again during the middle of the radiotherapy. RESULTS: Twelve patients with advanced squamous cell lesions were enrolled and 11 were observed to have complete clinical remissions. Seven patients remained clinically free of local disease at the time of death or most recent follow-up. Acute toxicities, usually hematologic or mucosal, were severe and all patients required treatment modifications and considerable supportive care. CONCLUSIONS: Although a high rate of response was achieved using this regimen, the toxicities are prohibitive. The kinetic profile of IdUrd incorporation suggests the need for future studies using repetitive short courses of IdUrd.

Adult↗

PubMed1997

Upper airway obstruction in the rheumatic diseases.

Upper airway obstruction is a potentially life-threatening event that can be managed effectively when promptly recognized and treated. For this reason, all patients who are suspected of having disease affecting the larynx or trachea should be regarded as having a potentially compromised airway until proven otherwise. Although clinically significant upper airway diseases occur infrequently in rheumatic disorders, these manifestations should be considered in such patients who present with upper airway symptomatology.

Algorithms↗

PubMed1997

Metastatic variants derived following in vivo tumor progression of an in vitro transformed squamous cell carcinoma line acquire a differential growth advantage requiring tumor-host interaction.

The purpose of this study was to develop an experimental model of squamous cell carcinoma that can be used to identify molecular and immunologic changes associated with primary events in malignant transformation, and those associated with metastatic tumor progression in the presence of host homeostatic and immunologic factors. Metastatic variants were derived following in vivo tumor progression of the in vitro transformed squamous cell carcinoma line Pam 212. The parental and metastatic cell lines exhibited similar morphologic features and molecular markers of an epithelial lineage, including an epithelial morphology in culture, cell surface expression of integrin alpha6beta4, and expression of mRNA of cytokeratins K6 and K14. When the growth and metastatic phenotype of the parental and reisolate cell lines was compared, the reisolate cell lines were found to exhibit a greater rate of growth and incidence of metastasis than the parental cell line when reimplanted in vivo. The difference in the growth rate of the parental cell line and the variants observed in vivo was not detected when growth of these lines was compared in vitro, suggesting that the growth advantage and selection of these variants requires tumor-host interaction. The metastatic variants exhibited a similar growth advantage in normal immunocompetent and SCID Balb/c mice, indicating that the growth advantage in vivo is not due to T or B lymphocyte-dependent immune factor(s). We conclude that metastatic variants derived following in vivo tumor progression of an in vitro transformed squamous cell carcinoma line exhibit a differential growth advantage in vivo that requires the host environment. Comparison of these in vitro transformed and in vivo derived metastatic variant cell lines with phenotypic differences in growth and metastasis should prove useful for dissecting the role of tumor and host factor(s) in malignant transformation and metastatic tumor progression of squamous cell carcinoma.

Animals↗

PubMed1997

Genes differentially expressed with malignant transformation and metastatic tumor progression of murine squamous cell carcinoma.

Molecular changes occurring with tumor formation and metastasis need to be identified in order to define novel markers and targets for chemoprevention and therapy. Cell lines from a multistage model of murine squamous cell carcinoma were analyzed for differences in gene expression using mRNA differential display. mRNA was isolated from primary keratinocytes, an in vitro transformed keratinocyte line (Pam 212), and three metastatic cell lines derived from Pam 212 following tumor progression in vivo. cDNA was synthesized by reverse transcription and amplified by PCR using 72 primer combinations to screen and compare approximately 3,600 sequences. Five cDNAs with a differential expression pattern confirmed by Northern blot analysis were cloned and sequenced, revealing homology with known genes. The gene encoding tropomyosin alpha was preferentially expressed in primary keratinocytes; genes for tyrosine kinase Yes-associated protein (YAP65) and ribosomal protein L18a were preferentially expressed in transformed and metastatic tumor cell lines; and genes for the Gro-alpha family cytokine KC and antigen Sp17 exhibited increased expression in the three metastatic cell lines. The structure and function of the genes identified suggest that they may possibly be linked to cell shape and motility, signal transduction, protein synthesis, growth, granulocyte chemotaxis, and angiogenesis. This study demonstrates the ability of mRNA differential display to detect altered gene expression in this tumor progression model of murine squamous cell carcinoma, and the potential usefulness of this approach for identification of candidate genes as chemoprevention markers and targets.

Amino Acid Sequence↗

PubMed1996

Immunodominance deters the response to other tumor antigens thereby favoring escape: prevention by vaccination with tumor variants selected with cloned cytolytic T cells in vitro.

Variant cancer cells which arise from the parent tumor during tumor progression can escape immunity but retain antigens. We have mixed highly immunogenic (A+B+) murine parental cancer cells with less immunogenic (A-B+) variant cancer cells to construct a model of a cancer containing escape variants. When such mixtures of cancer cells were injected into normal mice, the variant cells grew out because immune responsiveness to the B antigen on the variant was hindered by dominance of the A antigen on the surrounding parental tumor cells. However, A-B+ variant cells inoculated alone at a separate site induced B specific cytolytic T cells and were rejected. Moreover, mice immunized with A-B+ cells rejected a challenge which contained a mixture of variant and parental cancer cells, while immunization with A+B+ cells was ineffective. Thus, variant tumor cells selected from parental tumor cells by cytolytic T cells in vitro can be used to induce protective immunity against variants expected to escape tumor immunity in vivo. The immunodominance of the A antigen may be related to its ability to induce a much more rapid CTL response than the B antigen, since we show in another model that the pre-existence of a CTL response to one antigen prevented the subsequent induction of CTL to another antigen injected at the same site, even if both antigens were equally efficient at inducing CTL. These results indicate that immunodominance can affect strong as well as weak antigens. Vaccination with individual antigens at separate sites rather than with multiple antigens at one site may, therefore, be needed to prevent tumor escape and tumor recurrence or to counteract infectious diseases.

Animals↗

PubMed1995

Increase in suprabasilar integrin adhesion molecule expression in human epidermal neoplasms accompanies increased proliferation occurring with immortalization and tumor progression.

In a previous prospective study of 80 patients with squamous cell carcinoma of the upper aerodigestive tract, a progressive increase in expression of the integrin cell adhesion molecule alpha 6 beta 4 in suprabasilar cell layers of the tumor parenchyma was associated with an increase in early recurrence after therapy. In this study, we determined the relationship of the altered expression pattern of the integrin to changes occurring during benign, invasive, or metastatic stages of tumor development. Suprabasilar expression of integrin alpha 6 beta 4 appeared with neoplastic transformation in benign squamous papillomas, but homogeneous expression occurred more frequently in the parenchyma of primary and metastatic squamous cell carcinomas. The variation in the extent of suprabasilar integrin expression among the tumors corresponded to the variation in the population undergoing proliferation as determined by two independent markers of proliferation. Integrin expression was quantified in primary, HPV 16 DNA-immortalized, and v-ki-ras oncogene-transformed keratinocytes, and the pattern of expression was compared with cell cycle progression. Primary keratinocyte lines showed a bimodal distribution of integrin expression, with one population showing decreased integrin expression, cell size, and a block of cell cycle progression consistent with differentiation, whereas another population exhibited high integrin expression and full progression through the cell cycle, consistent with proliferation. HPV-immortalized and v-ki-ras-transformed cell lines undergoing continuous proliferation exhibited uniformly strong integrin expression, which was similar in intensity to that observed in the proliferating population of normal keratinocytes. Similar increases in expression of two additional integrins, alpha 2 beta 1 and alpha 3 beta 1, occurred along with integrin alpha 6 beta 4 in tissue specimens and cell lines derived from neoplasms. Thus, epidermal neoplasms display an increase in a population of cells exhibiting constitutive expression of a repertoire of integrins, which is similar to that found transiently in the acute phase of epidermal wound healing, a physiological response in which hyperproliferation, retention of multiple layers of proliferating cells, and migration occur. The association of a progressive increase in suprabasilar expression of these integrins with early tumor recurrence and advanced neoplasia suggests that constitutive expression and function of the same repertoire of integrins may be advantageous, rather than sufficient, for tumor progression.

Antigens, Surface↗

PubMed1993

Blood group antigens and integrins as biomarkers in head and neck cancer: is aberrant tyrosine phosphorylation the cause of altered alpha 6 beta 4 integrin expression?

Head and neck cancer is a capricious disease that varies greatly in its clinical behavior. The development of biomarkers that can distinguish between biologically aggressive and indolent tumors has been a long term goal of our laboratories. Predictive markers applicable to biopsy specimens should facilitate clinical management through early identification of patients at greatest risk for early relapse or metastatic spread. Two prominent cell surface markers that we identified by raising monoclonal antibodies to squamous cell carcinomas are blood group antigens and the A9 antigen/alpha 6 beta 4 integrin. Both of these markers are abnormally displayed in squamous cancers of the head and neck and serve as indicators of early relapse. Loss of blood group antigen expression is a stronger single indicator than is overexpression of the alpha 6 beta 4 integrin. However, use of both markers together is a stronger predictive indicator than is either alone. We know little about the function of the blood group antigens in squamous cells except that the mature antigens are associated with differentiation. Similarly, the function of the alpha 6 beta 4 integrin is also not fully understood. Integrin alpha 6 beta 4 is thought to serve as an extracellular matrix receptor, but its ligand has not been confirmed. In resting epithelium, the alpha 6 beta 4 integrin is polarized to the basal aspect of the basal cell as a component of the hemidesmosome, the anchoring structures of the epithelia. This basal polarization is lost in migrating normal squamous cells and squamous carcinomas. Tyrosine phosphorylation of the beta 4 subunit is absent or greatly reduced in malignant cells and this may be a critical signal for subcellular localization of alpha 6 beta 4 and cell anchoring. On the basis of our current experimental results, we postulate that tyrosine phosphorylation of the beta 4 subunit is a reversible signal that regulates cell migration in normal and malignant cells, and may therefore be an important initial event in the metastatic cascade.

ABO Blood-Group System↗

PubMed1993

The monoclonal antibody BQ16 identifies the alpha 6 beta 4 integrin on bladder cancer.

Monoclonal antibody BQ16, raised against UM-UC-9, a human bladder cancer cell line, exhibited strong reactivity with most bladder carcinoma tissue samples and cell lines. In normal urothelium, BQ16 stained only the basal surface of urothelial cells at the junction with the lamina propria. BQ16 immunoprecipitated two protein bands of approximately 140 and 180 kDa (under non-reducing conditions), while on Western blots, BQ16 identified only the 140 kDa protein indicating that BQ16 binds to one chain of a dimeric protein complex. The dimeric structure, molecular size, and basal orientation of the BQ16 antigen prompted a comparison with the alpha 6 beta 4 integrin identified by monoclonal antibody UM-A9. In most tissues BQ16 and UM-A9 produced identical staining patterns. However, normal lymphocytes and certain bladder cancer cell lines were BQ16 positive but failed to react with UM-A9, indicating that the BQ16 and UM-A9 epitopes can be expressed independently. Pulse-chase immunoprecipitation experiments showed that the alpha 6 subunit was more prominent in early BQ16 precipitates and the beta 4 subunit was more prominent in early UM-A9 precipitates. Furthermore, preclearing cell extracts with the anti-alpha 6 antibody GoH3 removed all BQ16 reactivity and in UM-A9-negative, BQ16-positive cells, BQ16 precipitated the alpha 6 beta 1 complex. We conclude that BQ16 identifies the alpha 6 integrin subunit and that alpha 6 beta 4 integrin is strongly expressed in most bladder cancers.

Antibodies, Monoclonal↗

PubMed1992

Overexpression of the A9 antigen/alpha 6 beta 4 integrin in head and neck cancer.

A tumor-associated antigen detected by monoclonal antibody UM-A9 raised against a cultured cell line from a patient with an aggressive SCC of the oral cavity has been defined. The A9 antigen is abnormally expressed in squamous cancers, with loss of basal polarization and increased intensity of expression distinguishing malignant from normal cells. A minority of cultured SCC cell lines and about one third of fresh tumors exhibit polarized A9 expression. The increased intensity and loss of polarized expression of A9 antigen in recurrent and metastatic tumor cell lines when compared with primary or early tumor cell lines from the same patients indicated an association of altered expression with tumor progression. When A9 expression was evaluated in frozen tumor sections, three patterns of expression representing increasing intensity and loss of polarization were observed. Patients whose tumors exhibited the most intense A9 antigen expression had a higher rate of early relapse than patients whose tumors exhibited low intensity and polar expression. Loss of blood group antigen expression was also associated with poor prognosis, and together high A9 antigen expression and loss of blood group defined a group of patients at high risk of early relapse. The A9 antigen is immunologically and biochemically identical to the alpha 6 beta 4 integrin. The association of high expression of the A9/alpha 6 beta 4 integrin as a prognostic factor is supported by similar findings with a mouse model system. The mouse tumor-associated antigen, TSP-180, which is also an alpha 6 beta 4 integrin, distinguishes highly metastatic tumor cells from nonmetastatic variants of the same tumor line. In SCC, the alpha 6 beta 4 integrin contributes to attachment to laminin since anti-alpha 6 subunit specific antibody blocks cell attachment and only the beta 4 subunit is found in association with the alpha 6 subunit in these cells. Similar findings were obtained in colon carcinomas. Antibodies and peptides that block laminin attachment may lead to the development of antimetastatic agents for squamous carcinomas. The beta 4 subunit is unique from other integrins in that it has an unusually long cytoplasmic domain, the function of which is not known. The beta 4 subunit is heavily phosphorylated under conditions that favor anchoring and terminal differentiation in normal keratinocytes. Paradoxically the beta 4 subunit is also heavily phosphorylated in tumor cells, which are highly migratory and immortalized.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Neoplasm↗

PubMed1992

Regulation of expression and phosphorylation of A9/alpha 6 beta 4 integrin in normal and neoplastic keratinocytes.

The A9 antigen is a basement membrane antigen of normal squamous epithelial cells that is strongly expressed in many squamous carcinomas. High expression of this antigen is associated with early relapse in squamous cell carcinomas of the head and neck. We now know that the A9 antigen is structurally, immunologically, and functionally similar to the alpha 6 beta 4 integrin that has been shown to be linked to metastatic behavior in murine tumor models. The alpha 6 and beta 4 genes have been cloned and sequenced, and a model has been constructed from the deduced amino acid composition. In this study we present a hypothetical model and use it to design experiments to assess the factors that influence the expression of the A9/alpha 6 beta 4 integrin in normal and malignant keratinocytes. High calcium induces down regulation of A9/alpha 6 beta 4 antigen in normal but not malignant keratinocytes within 24 hours. Although calcium can down-regulate beta 4 message in tumor cells in the absence of epidermal growth factor (EGF), transcription of beta 4 increased in the tumor cells under the conditions we used for assessing antigen expression (calcium plus EGF). Retinoic acid also stimulated transcription of beta 4 in tumor cells, but this was partially inhibited by the presence of high calcium. Phosphorylation of the beta 4 chain was stimulated by epidermal growth factor and calcium in normal keratinocytes, but in the malignant cells phosphorylation was constant regardless of the culture conditions. Our results indicate that high expression of the alpha 6 beta 4 integrin is associated with conditions that favor migration and undifferentiated proliferation of normal keratinocytes and that malignant keratinocytes differ from normal keratinocytes by constitutive phosphorylation of beta 4 and by failure to downregulate beta 4 transcription in response to calcium in the presence of EGF.

Antigens, Neoplasm↗

PubMed1991

The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin alpha 6 beta 4.

We previously reported that altered expression of the A9 antigen (defined by monoclonal antibody UM-A9) is a predictive marker of early recurrence and progression of squamous cell carcinoma (SCC). In normal squamous cells A9 expression is limited to the site of contact with the basement membrane in vivo and the culture surface in vitro, whereas aggressive SCCs exhibit loss of polarity and increased intensity of A9 expression. The potential relationship of the A9 antigen to structures known to be involved in cell adhesion was analyzed by immunobiochemical and cell adhesion assays. UM-A9 precipitates a complex of protein chains reminiscent of the alpha and beta heterodimer glycoproteins that characterize the integrin family of extracellular matrix receptors. Proteins were isolated from A9-positive cells using UM-A9 and well-defined antibodies specific for integrin alpha and beta chains. UM-A9, anti-alpha 6, and anti-beta 4 monoclonal antibodies (mAbs) all precipitated proteins with comparable electrophoretic mobilities. Furthermore, UM-A9 mAb precleared the SCC alpha 6 beta 4 integrin complex isolated with anti-alpha 6 or anti-beta 4 mAbs but not that isolated by anti-beta 1 mAb. The isoelectric points of the A9 complex chains were consistent with those reported for alpha 6 and beta 4. Three of the polypeptide chains (140, 175, and 205 kDa) precipitated by UM-A9 showed peptide homology to one another and to the beta 4 chain precipitated by mAb 439-9B. The A9/alpha 6 subunit is composed of 125- and 30-kDa chains and was distinguished from beta 4 and beta 1 chains by its peptide map and isoelectric point. UM-A9 binds to an epitope common to the beta 4 subunits since in pulse-chase analysis the beta 4 species are precipitated at an early time point, whereas detection of alpha-subunit synthesis is detected during assembly of the mature complex. Immunoprecipitation and preclearing experiments demonstrated that in SCC the alpha 6 subunit is associated primarily with the beta 4 species and not with the 130-kDa beta 1 subunit. In cell adhesion assays on extracellular matrix proteins, the alpha 6-specific GoH3 mAb inhibited binding of SCC to laminin, suggesting that alpha 6 beta 4 may function as a laminin receptor in SCC. These data and our prior observations showing an association between altered A9 expression and early recurrence in SCC provide the first evidence that altered expression of alpha 6 beta 4 integrin is associated with the clinical behavior of human squamous cell carcinomas.

Antigens, Neoplasm↗