Expression of Po66-CBP, a galectin-8, in different types of primary and secondary broncho-pulmonary tumors.

The monoclonal antibody Po66 is an IgG1 immunoglobulin isolated from a human bronchial squamous carcinoma and directed against a carbohydrate binding protein, Po66-CBP, belonging to the galectin family involved in neoplastic processes. This Po66 antibody has been shown to be useful for immunoscintigraphic detection of squamous cell carcinoma metastasis. We examined the expression of Po66-CBP in a wider range of primary or secondary malignant tumors of the lung of various histological types. We studied 52 specimens of broncho-pulmonary tumors including 41 primary squamous, glandular or neuro-endocrine tumors and 11 secondary tumors of glandular, connective tissue, melanocytic or germinal origin as well as 9 extra-pulmonary primary tumors with histological types similar to lung metastases. An immunohistochemical study was performed using an amplification system on paraffin-embedded sections. All histological types were positive for Po66 antibody, the cell origin giving no influence on the expression of Po66-CBP. There was however a relation between Po66-CBP expression and the degree of differentiation notably for squamous cell cancer and neuro-endocrine tumors. The metastatic character of the tumor tissue did not affect Po66-CBP expression.

Protein based microarrays: a tool for probing the proteome of cancer cells and tissues.

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.

A database of protein expression in lung cancer.

We have developed a comprehensive approach to identifying molecular changes in lung cancer that includes both genomic and proteomic analyses. The related effort has produced a large amount of data pertaining to gene expression at the RNA and protein levels. As a result, we have constructed a database that contains protein expression data on lung cancer as well as other relevant data including DNA microarray derived data. A large number of proteins that are expressed in different types of lung cancer have been identified and have been correlated with the expression measures for their corresponding genes at the RNA level. The database is intended to facilitate our effort at developing novel classification schemes for lung cancer and the identification of novel markers for early diagnosis.

Proteomics-based identification of protein gene product 9.5 as a tumor antigen that induces a humoral immune response in lung cancer.

We used a proteomic approach to identify proteins that commonly induce an antibody response in lung cancer. Sera from 64 newly diagnosed patients with lung cancer, 99 patients with other types of cancer, and 71 noncancer controls were analyzed for antibody-based reactivity against lung adenocarcinoma proteins resolved by two-dimensional PAGE. Unlike controls, autoantibodies against a protein identified by mass spectrometry as protein gene product 9.5 (PGP 9.5) were detected in sera from 9 of 64 patients with lung cancer. Circulating PGP 9.5 antigen was detected in sera from two additional patients with lung cancer, without detectable PGP 9.5 autoantibodies. PGP 9.5 is a neurospecific polypeptide previously proposed as a marker for non-small cell lung cancer, based on its expression in tumor tissue. Using A549 lung adenocarcinoma cell line, we have demonstrated that PGP 9.5 was present at the cell surface, as well as secreted. Thus, the findings of PGP 9.5 antigen and/or antibodies in serum of patients with lung cancer suggest that PGP 9.5 may have utility in lung cancer screening and diagnosis.

Two messenger RNAs and five isoforms for Po66-CBP, a galectin-8 homolog in a human lung carcinoma cell line.

Galectins are animal proteins which specifically bind beta-D-galactoside residues and their specific cellular function is not yet clearly established. However, these proteins seem to play a role in neoplastic transformations. Po66 is a murine monoclonal antibody directed against a protein from human lung carcinoma, Po66 Carbohydrate-Binding-Protein (Po66-CBP), which belongs to the galectin-8 family. Our results show that the Po66-CBP gene generates five transcripts by alternative splicing, which could give rise to five proteins: two proteins belong to the tandemly repeated galectin family and three belong to the single carbohydrate recognition domain galectins. All these proteins are encoded by a unique gene located in 1q42. Experiments carried out by reverse transcriptase-polymerase chain reaction show that the levels of expression of these five galectin-8 isoforms are variable during the culture time in SK-MES-1, a human lung squamous carcinoma cell line. Cancer Genome Anatomy Project database analysis confirms the presence of Po66-CBP in lung cancer and its absence in healthy lung.

Galectin-8: a complex sub-family of galectins (Review).

Galectins are animal lectins, that can specifically bind beta-galactosides. Twelve galectins have been described in vertebrates, belonging to three different groups: prototype, tandem-repeat and chimeric. These proteins seem to be involved in cellular interactions and neoplastic transformations. We present an overview of a particular galectin member: galectin-8. This galectin, which has been intensively studied over the last six years, presents a particular type of gene regulation. It is widely expressed in tumoral tissues and seems to be involved in integrin-like cell interactions. Studies show that the LGALS8 gene encodes for almost seven mRNAs by alternative splicing pathways and various polyadenylation sites. These mRNAs could encode for six isoforms of galectin-8, of which three belong to the tandem-repeat galectin group (with two carbohydrate binding domains) and the three others to the prototype group (one carbohydrate binding domain). All these isoforms seem to be differentially expressed in various tumoral cells. This untypical galectin-8 subfamily seems to have a complex expression regulation, that could be involved in cancer phenomena.

An immune response manifested by the common occurrence of annexins I and II autoantibodies and high circulating levels of IL-6 in lung cancer.

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. The purpose of this study was to identify proteins that commonly induce a humoral response in lung cancer by using a proteomic approach and to investigate biological processes that may be associated with the development of autoantibodies. Aliquots of solubilized proteins from a lung adenocarcinoma cell line (A549) and from lung tumors were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for primary antibodies. Sera from 54 newly diagnosed patients with lung cancer and 60 patients with other cancers and from 61 noncancer controls were analyzed. Sera from 60% of patients with lung adenocarcinoma and 33% of patients with squamous cell lung carcinoma but none of the noncancer controls exhibited IgG-based reactivity against proteins identified as glycosylated annexins I and/or II. Immunohistochemical analysis showed that annexin I was expressed diffusely in neoplastic cells in lung tumor tissues, whereas annexin II was predominant at the cell surface. Interestingly, IL-6 levels were significantly higher in sera of antibody-positive lung cancer patients compared with antibody-negative patients and controls. We conclude that an immune response manifested by annexins I and II autoantibodies occurs commonly in lung cancer and is associated with high circulating levels of an inflammatory cytokine. The proteomic approach we have implemented has utility for the development of serum-based assays for cancer diagnosis as we report in this paper on the discovery of antiannexins I and/or II in sera from patients with lung cancer.

Sodium butyrate induces growth inhibition and modulates galectin-8 expression in human lung carcinoma cells.

Galectins are animal lectins, which may play a role in neoplastic transformation. Po66-Carbohydrate Binding Protein (Po66-CBP) belongs to the galectin-8 family and is expressed in lung tumor cells but not in normal ones. Recent studies showed that galectin-8 could be used for human lung squamous cell carcinoma radioimmunotherapy. To optimize this method of treatment, we attempted to increase galectin-8 expression in human lung tumor cells. A human lung squamous (SK-MES-1) or adeno (A 549) carcinoma cell line was grown with or without sodium butyrate. Cell growth, morphology, transcriptional, expression translational expression and cellular localization of galectin-8 were studied. 3 mM of sodium butyrate inhibited the two cell lines' growth after 48 hours of treatment, but only in SK-MES-1 cells galectin-8 expression is modulated without any secretion and cellular localization modifications, apoptosis or necrosis. Sodium butyrate could be an interesting tool in optimizing the radioimmunotherapy of human lung squamous carcinoma, but not of adenocarcinoma.

Immunohistochemical expression of the intracellular component of galectin-8 in squamous cell metaplasia of the bronchial epithelium in neoplastic and benign processes.

Specified galectins are known to play a role in regulating cell proliferation, differentiation, adhesion and migration. Po66, a mouse IgG1 monoclonal antibody produced by immunization against squamous cell cancer, reacts against a carbohydrate-binding protein (Po66-CBP), recently shown to be a member of the galectin family with a strong homology with galectin-8 (PCTA-1), identified as a human tumor-associated antigen. We studied Po66 in squamous metaplasia of the bronchi in order to determine whether it could be specifically involved in neoplastic conditions and if so, if it would be helpful in distinguishing metaplasia at risk of cancer. Twenty-eight formalin-fixed, paraffin-embedded archival tissues of 17 metaplasias with SCC, 3 metaplasia with distant neoplastic disease and 8 metaplasias with an inflammatory process, were immunostained using a streptavidin biotin peroxydase method. The squamous metaplasias were positively stained in non-neoplastic disease as well as in neoplastic processes. Expression was also observed in stromal and normal cells. Po66-CBP was not associated with a pre-neoplastic character. We discussed the expression of this intra-cellular component of galectin-8 according to the functions of galectins in cellular differentiation, host reaction against tumor, and inflammation.

A proteomic approach to the identification of lung cancer markers.

We have developed a comprehensive approach to the identification of protein markers in lung cancer that includes profiling of tumor tissue and cell lines as well as the analysis of serum for autoantibodies to lung tumor antigens. A large number of proteins that are differentially expressed in the major subtypes of lung cancer have been identified by mass spectrometry. A database of protein expression in lung cancer and other types of cancer has been constructed that integrates two-dimensional gel profiles, mass spectrometry data, quantitative protein data and gene expression data at the RNA level, that serves as a resource for biomarker identification. Analysis of the serological response in lung cancer has led to the identification of novel markers detectable in serum of lung cancer patients at the time of diagnosis. The proteomic approach is likely to yield novel classification schemes and novel markers for early diagnosis of lung cancer.

[Vertebrate galectins: structure and function, role in tumoral process]. / Les galectines animales: structure et fonctions, rôle dans les processus tumoraux.

Galectins are proteins structurally related to the lectin family. They share, with lectins, the ability to bind carbohydrate residues. Galectins are suspected to mediate several biological functions such as embryonic development growth, immune response and apoptosis. Their role is similar to that of adhesion molecules in cell to cell or to matrix interactions. Their contribution to human carcinogenesis has been suggested from experimental studies. In clinical research, they could be used as a differentiation marker, particularly in thyroid carcinomas and in certain lymphomas.

Expression of Po66-CBP, a type-8 galectin, in different healthy, tumoral and peritumoral tissues.

UNLABELLED: A monoclonal antibody, Po66, recognized an antigen named Po66 carbohydrate binding protein (PO66-CBP), which was homologous to the galectin-8 protein. Two additional isoforms previously not described were characterized. The aim of this study was to compare the expression of Po66-CBP and its isoforms in different healthy, tumoral and peritumoral tissues and at last to determine the localization of the protein in tumors and distant tissues. MATERIALS AND METHODS: Reverse transcriptase PCR of Po66-CBP was performed on total RNA extract from eleven healthy and eleven tumoral and peritumoral tissue specimens. Antibody Po66 was used to localize the protein in the tumors and distant tissues by an immunohistochemistry method. RESULTS: Po66-CBP was expressed by half of the healthy tissues. One of the isoforms, the last often present in healthy tissues, was found in all tumoral and peritumoral tissues studied. Immunohistochemistry evidenced a gradient of protein expression in normal cells depending on the vicinity of tumoral tissue. Po66-CBP expression was modified in cancerous tissue, suggesting the implication of galectins in carcinogenesis.

The uptake of a monoclonal antibody (Po66) in lung tumour cell aggregates involves a phagocytose-like phenomenon.

The efficacy of radioimmunotherapy with radiolabelled monoclonal antibody (Mab) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. Multicellular spheroids (MTS) of lung cell carcinoma was investigated to study cellular and subcellular distribution of a Mab Po66 labelled with I-125. We have shown that the incorporation of Po66 in MTS regularly increases during 3 days while Py, a non specific Mab, remains low. The distribution of radiolabelled studied by light and electron microscopy autoradiography have demonstrated that Po66 first localized on extra cellular debris, is then phagocyted and observed in the cytoplasm of viable cells. This mechanism of penetration and distribution of Mab Po66 is a new and interesting phenomenon, and emphasizes contribution of transmission electron microscopy in nuclear medicine.

Treatment of human lung carcinoma xenografts with a combination of 131I-labelled monoclonal antibody Po66 and doxorubicin.

Po66, a mouse monoclonal antibody, is directed against an intracytoplasmic antigen present in human lung squamous cell carcinoma cells. In previous work it was found that the co-administration of 125I-radiolabelled Po66 and doxorubicin strongly enhanced the uptake of radioactivity by the tumour. The present-work was designed to evaluate, in a tumour-bearing mouse model of lung carcinoma, the ability of 131I-labelled Po66 to retard tumour growth when injected alone, or in combination with doxorubicin (8 mg kg-1 at 1-week intervals). A single dose of 550 microCi 131I-Po66 alone had no effect on tumour growth, whereas three fractionated doses of 250 microCi 131I-Po66 decreased it over two doubling times from 14.5 +/- 1.5 days for untreated control mice to 24.8 +/- 2.7 days. Mice treated with doxorubicin alone had a double tumour doubling time of 22.6 +/- 4.9 days, compared to 35.2 +/- 2.9 days (1.55-fold increase) in mice treated with doxorubicin and a single dose of 550 microCi 131I-Po66. Doxorubicin combined with three fractionated doses of 250 microCi 131I-Po66 provoked a twofold decrease in tumour growth compared to mice treated with doxorubicin alone. The administration of fractionated doses of 131I-Po66 simultaneously with doxorubicin resulted in a highly delayed mortality, which was not observed when 131I-Po66 was administered after doxorubicin. Thus, in a non-small-cell lung tumour model, a 131I-radiolabelled monoclonal antibody, directed against an intracellular antigen, significantly potentiated the effect of chemotherapy. Such a therapeutic approach could be used as an adjuvant therapy and improve the effect of chemotherapy on distant small metastases.

Purification of a tumoral marker recognized by monoclonal antibody Po66 and associated with human lung squamous cell carcinoma.

Monoclonal antibody (MAb) Po66, a murine IgG1, was raised by immunization against human lung squamous cell carcinoma. When injected intravenously, Po66 showed prolonged retention in the tumor. It recognized an intracellular antigen. The human lung squamous carcinoma cell line SK-MES-1 expresses the antigen recognized by MAb Po66 and was used as a source of biological material for its purification. The SK-MES-1 cell line was labeled in culture with [35S]methionine and its lysate was immunoprecipitated with Po66 immobilized on Protein G-Sepharose. The precipitate contained three proteins (47, 50 and 69 kDa) absent in the controls. The 69 kDa polypeptide was further purified by anion exchange and immunoaffinity chromatographies. To date, no other tumor marker expressed in non-small cell lung cancer with these characteristics has been described and as such this marker is interesting for future use in immunotherapy and in diagnosis.

Monoclonal antibody Po66 uptake by human lung tumours implanted in nude mice: effect of co-administration with doxorubicin.

The efficacy of radioimmunotherapy of tumours with radiolabelled monoclonal antibodies (MAbs) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. In the case of MAbs directed against intracellular antigens, increasing the permeability of the cytoplasmic membrane may augment the bioavailability of the antigen for the antibody. This raises the question whether the induction of tumour necrosis by chemotherapy can enhance the tumour uptake of radiolabelled monoclonal antibodies. In this work, the effect of doxorubicin on the biodistribution of Po66, an MAb directed against an intracellular antigen, was studied in nude mice grafted with the human non-small-cell lung carcinoma cell line SK-MES-1. After injection on day 0 of 125I-labelled Po66, tumour radioactivity increased up to days 3-5, and then remained unchanged to day 14. The combined administration of 125I-labelled Po66 with 8 mg kg-1 doxorubicin, in two doses separated by 7 days, doubled the radioactivity retained by the tumour. Histological and historadiographic analysis showed, however, that the drug induced cellular damage. In the absence of doxorubicin, the accumulation of Po66 was restricted to some necrotic areas, whereas with doxorubicin the necrosis was more extensive and the antibody more evenly distributed. These results suggest that chemotherapy and immunoradiotherapy combined would enhance tumour uptake of radioisotope and promote more homogenous distribution of the radiolabelled MAb. This would promote eradication of the remaining drug-resistant cells in tumours.