The receptor for advanced glycosylation endproducts (RAGE) is abundant at both the transcriptional and translational level in normal lung but is not expressed in non-small cell lung cancer (NSCLC). In order to determine whether sequence variations might be responsible for the inactivation of RAGE in NSCLC, we investigated the RAGE gene in primary NSCLCs and in the corresponding normal tissues of nine patients. Although sequence analysis revealed no somatic, tumor-associated mutations, six novel sequence variants were identified: T→A in the promoter region 388 bp upstream of the start codon: T→A in exon 1 (Ala2Ala), C→G in exon 3 (Val89Val), C→T in intron 6, G→C and C→G in exon 10 (Arg365Ser and Arg369Gly). In addition, we detected a 63 bp deletion in the promoter region (358–421 bp upstream of the start codon) in one NSCLC patient. The T→A transversion in the promoter region was detected in three of nine patients. Further analysis of this polymorphic locus in 54 NSCLC patients and 59 non-cancer controls revealed a significant difference in the genotype distribution between NSCLC patients and controls. Interestingly, the AA genotype was more common in NSCLC patients (20.8%) than in controls (3.5%). The cumulative occurrence of the AA variant in NSCLC suggests that this genotype is a putative risk factor for NSCLC development.

Human SPARC-like 1 (SPARCL1), also known as MAST9 or hevin, is a member of the SPARC protein family. Originally we identified SPARCL1 as one of the genes down regulated in human non-small cell lung cancer (NSCLC). Recent reports indicate that the down regulation of SPARCL1 also occurs in prostate and colon carcinomas, suggesting that SPARCL1 inactivation is a common event not only in NSCLCs but also in other tumors of epithelial origin. In the present work we report the cloning and mapping of the genomic locus of human SPARCL1. Using fluorescence in situ hybridization analysis, SPARCL1 was localized to chromosome 4q22-25, a region often deleted in human cancers. Furthermore, we show that the intron/exon organization of the human SPARCL1 gene is similar to its murine homologue SC1. SPARCL1 contains 11 exons and 10 introns which span approximately 47 kb of the genome. We also sequenced the 5'-flanking region of the human SPARCL1 gene containing 2.4 kb of the putative promoter region. The data presented herein are a prerequisite for deletion/mutation analysis of the SPARCL1 gene in tumors. In addition, knowledge of the SPARCL1 promoter sequence allows to investigate the regulation of SPARCL1 expression on the transcriptional level. Taken together our results will help to clarify the function of SPARCL1 in tumor formation.

The Tet gene expression system, that allows tightly controlled gene expression in response to doxycycline, was applied to analyze the influence of the receptor for advanced glycosylation endproducts (RAGE) on the growth of 293 cells in semisolid medium. Establishing a Tet-On gene expression system involves two consecutive stable transfections. Here, we describe an alternative procedure to obtain a Tet-On gene expression system in a single transfection step for the use in tumor biology. The plasmids necessary for the regulated expression of RAGE together with the selectable marker plasmid were cotransfected in a molar ratio of 6:1. After aminoglycoside selection, 29 clones were analyzed using PCR revealing 8 colonies to be double stably transformed. Subsequent Western blot analysis showed inducible expression in 7 cell lines. Applying the one step protocol, the entire Tet-On expression system could be completed in half of the time required for the original two step method. The generated 293 double stable cells were used in the clonogenic assay for the testing of the tumor suppressive potential of RAGE.

A previous study of 48 primary non-small cell lung carcinomas (NSCLC) for allelic loss at five polymorphic chromosome 11p loci indicated regions of allelic deletion for both 11p13 and 11p15. To further delineate the extent of this deletion 28 NSCLCs were examined by high resolution deletion mapping for allelic loss at 11p13.95% (18/28) of the informative cases displayed allelic loss at the catalase gene locus (CAT), 64% (18/28) at D11S935 and D11S941E, respectively, and 23% (6/26) at D11S907. The minimal region of deletion is bordered by CAT and D11S935 and spans about 3 cM. The relationship between allelic loss within this chromosomal region, the presence of a putative predisposition locus and the pathogenetics of NSCLC are discussed.

In the search for genes down-regulated in non-small cell lung cancer (NSCLC), we have identified a cDNA fragment, termed MAST9. Cloning, sequencing, and characterization of the full-length MAST9 cDNA revealed that the entire 2808 nucleotide sequence had an open reading frame of 1992 nucleotides encoding a Mr 75,000 protein. Sequence analysis disclosed a striking homology to SPARC, known to be involved in tumorigenesis. The recently identified "Hevin" cDNA isolated from high endothelial venules is identical to MAST9. Using Northern and Western blot analysis, we showed that MAST9 was down-regulated in the tumor samples of nine non-small cell lung carcinoma patients. Furthermore, we demonstrated that both the bacterially expressed and the endogenous MAST9 proteins form homodimers. The lack of expression in non-small cell lung carcinomas and its homology to SPARC suggest a putative role of MAST9 in lung tumor formation.

The receptor for advanced glycosylated end products (RAGE), a member of the immunoglobulin superfamily, was one of the cDNA subtraction clones that was found to be differentially expressed in human lung and the corresponding tumor tissue. In nine additional matched normal/tumor pairs, a strongly reduced or missing expression, not only on a transcriptional level but also on a protein level, was demonstrated in the non-small cell lung carcinoma tissue. Because amphoterin, a physiological ligand of RAGE that is highly expressed in the lung, mediates cell differentiation via RAGE, a down-regulation of the receptor may be considered as a critical step in lung tumor formation. Furthermore, we determined the complete reading frame of RAGE.

Eleven non-invasive and 24 invasive transitional cell bladder cancers were analysed for molecular alterations to the p53 gene and nuclear accumulation of the p53 protein. 9% (1/11) of non-invasive rumours and 21% (5/24) of invasive tumours revealed nuclear accumulation in more than 50% of the tumour cells. PCR analysis of D17S30 showed loss of heterozygosity (LOH) in invasive tumours (3/24; 12%). Two invasive tumours harboured point mutations in exon 6 and exon 7, respectively (8%). Our results indicate that p53 protein overexpression correlates with tumour progression, p53 gene mutations and LOH detected by PCR.

Using a magnet-assisted subtraction technique, 17 complementary DNA (cDNA) clones were isolated that were expressed in the normal lung but were decreased or lost in the corresponding tumor tissue of a nonsmall cell lung carcinoma patient. The lack of expression of six magnet-assisted subtraction technique cDNA clones in three additional non-small cell lung carcinoma cases indicates their possible relevance for non-small cell lung carcinoma. Two cDNA clones revealed homology to genes specifically expressed in lung, i.e., pulmonary surfactant-associated protein B and the receptor for advanced glycosylation end products of proteins. Three cDNA clones showed identity to cDNA sequences encoding calmodulin-like protein, glutamine synthetase, and cytoskeletal beta-actin. One cDNA clone is identical to a recently described human expressed sequence tag whose gene is still unknown.

BACKGROUND:Carcinogenesis, the formation of solid tumors, is now widely accepted to represent a multistep process. Several genetic events, activation of proto-oncogenes and inactivation of tumor suppressor genes, are involved.DESIGN:Review of the literature for evidence that the concept of multistep transformation has relevance also for the formation of low-grade lymphoproliferative diseases.RESULTS AND CONCLUSION:The common translocations in low-grade lymphoid tumors are probably early events, predominantly involved in the activation of oncogenes, leading to growth stimulation or prolonged cell survival. As a result 'monoclonal lymphoproliferative disorders of undetermined significance (MLDUS)' occur, undetermined, because some translocations may not always led to tumor formation. For progression to full malignancy, additional genetic events are required besides sequential selection of variant subpopulations within the neoplastic clone. Recent data indicate that mutations and deletions of putative tumor suppressor genes, including the P53 and retinoblastoma genes, are also involved in the progression of lymphoproliferative disorders. A list of lymphoproliferative diseases stressing this concept of multistep transformation is presented in this article.

Although the occurrence of bladder cancer is common, the molecular events underlying the pathogenesis of this cancer remain ill-defined. A loss of heterozygosity (LOH) at specific chromosomal loci may predispose individuals to the development of bladder cancer but this has not been examined in detail. Furthermore, the role that deletion or inactivation of putative tumour suppressor genes might play in the genesis of bladder cancer has not been established. In this study, allelic deletion analysis on the short arm of chromosome 17 of patients with primary bladder tumours failed to show deletion at 17p13 (0/7), a region known to contain the p53 tumour suppressor gene. Chromosome 11p15 showed allelic deletion at the IGF2 locus (2/7: 29%) and the PTH locus (1/11: 9%). However, no deletion was observed at the CALCA locus (0/6). LOH at 11p13, a region containing the Wilm's tumour suppressor gene (WT1), was also studied. Analysis of LOH at 11p13 showed deletion at the CAT locus (13/18: 72%), the ΔJ/D11S414 locus (5/15: 33%), the WT1 locus (7/14: 50%) and the FSHB locus (6/16: 38%). The significance of these findings is discussed.

Forty‐eight samples of primary non‐small‐cell lung cancer (nsclc) and normal tissue from the same patients were analyzed for allelic deletions on chromosome 11p. five polymorphic loci were assessed to determine the incidence of 11 p sequence deletions and to define hot‐spots of deletions. information was obtained from all patients in at least one locus. our data show that the deletions observed were not randomly scattered over the short arm of chromosome i i. rather, 2 hot‐spots of deletions were observed: one in the area of the genes for catalase and β‐fsh corresponding to band ii p13, the other close to the igf‐ii locus corresponding to band ii p15. a high incidence of loss of heterozygosity (loh) was found with the probe for catalase (21/29), a locus flanking the centromeric region of the wilms' tumor locus. most of the samples exhibiting loh of one or more of the alleles analyzed remained heterozygous for at least one other chromosome iip allele. furthermore, duplication of the intensity of the remaining allele was rarely observed. our results indicate that loh on the short arm of chromosome ii is a common event in nsclc and that the chromosomal region containing the wilms' tumor locus is most commonly involved.

Clearcut progress has been achieved in the treatment of lung cancer in the last decade. While small cell lung cancer has proven a highly chemosensitive tumor, only remissions of relatively short duration are obtained. The high relapse rate is probably due to the reappearance of chemoresistant subclones. In contrast, non small cell lung cancer is often chemoresistant from the outset, a fact which limits the therapeutic possibilities in these tumors. Different forms of chemoresistance are described and potential modalities of circumventing or reversing it are discussed. In recent years, greater insight into molecular mechanisms of tumor formation has been achieved. The role of activation or overexpression of oncogenes has been demonstrated. More recently the role of tumor suppressor gene inactivation has become evident. By means of restriction fragment length polymorphism (RFLP) a combination of gene deletions in various chromosomes has been demonstrated in lung cancer. The pattern of chromosomes involved appears to vary between small cell and non small cell lung cancer. It is to be hoped that these new insights will be translated into new treatment modalities for lung cancer.

Totally implantable catheters have greatly improved therapy and supportive care of tumor patients. Venous catheters can reduce the problem of maintaining reliable vascular access and the risk of cytotoxic drug extravasation. In addition, epidural or intrathecal catheter systems can result in an improved pain relief and in reduced side effect incidences in many patients suffering from severe cancer pain. The intraarterial and intraperitoneal catheters facilitate loco-regional chemotherapy. If used by an experienced team, most of these systems have a relatively low complication rate. The combination of such catheters with portable pumps does further facilitate the management of ambulatory tumor patients and will improve their quality of life.

While brain metastases from small cell lung cancer are a familiar problem, the incidence of brain metastases from non-small cell lung cancer, and their significance as the first tumor manifestation, has been underestimated. At the University Hospital, Basle, over one year, 7 (approximately 7%) of 102 patients with newly diagnosed non-small cell lung cancer had brain metastases as the first manifestation of systemic cancer. Three of the 7 patients were women with a mean age of 48 years. Initial symptoms were headaches, vertigo and vomiting, which prompted the diagnosis of brain metastases. In only 3 patients was the primary lung cancer diagnosed immediately after diagnosis of the brain metastases, while in the remaining 4 a period of up to 6 months elapsed. Bronchogenic cancer is the most frequent primary in patients presenting with brain metastases. Accordingly, in a patient with brain metastases from an unknown primary, bronchogenic cancer should be considered first and diagnostic tests aimed in that direction. This may obviate an extended and expensive diagnostic workup.

The bioreductive alkylating agent mitomycin C (mitomycin) has been shown to have greater activity under hypoxic than oxic conditions on murine cell lines such as the EMT-6 fibrosarcoma cell line. Solid tumors are known to contain hypoxic cells and are relatively resistant to ionizing radiation and some chemotherapeutic agents. We tested the cytotoxicity of mitomycin against fresh biopsies of human carcinomas under both hypoxic and oxic conditions in the human tumor clonogenic assay (HTCA). Additionally, we examined the metabolism of mitomycin by sonicates of the murine EMT-6 cells and the human WiDR colon carcinoma cells. We confirmed that under our clonogenic assay conditions the EMT-6 cell line was more sensitive to mitomycin under hypoxic than oxic conditions. Additionally, we established that EMT-6 cells also metabolize mitomycin at a more rapid rate under hypoxic than oxic conditions. However, these effects of hypoxia on mitomycin activity were not demonstrable for the human WiDR colon cancer cell line. In addition to these findings, the cytoxicity of mitomycin was either unchanged or reduced under hypoxic conditions for ten fresh human tumors tested for mitomycin sensitivity in HTCA. Based on these observations, we conclude that the potentiating effect of hypoxia on mitomycin metabolism and biological activity may be peculiar to the murine EMT-6 and S-180 cell lines and that mitomycin C is not likely to have differential efficacy against hypoxic human carcinoma cells.

T lymphocyte colony forming cells (TL-CFC) grown in agar in the presence of PHA were assayed for their capacity to induce or suppress polyclonal PWM dependent B lymphocyte differentiation into plasma cells. This was measured by identifying cells containing intracytoplasmatic immunoglobulins by direct immunofluorescence. To validate the helper and suppressor system used in this paper, the inductive capacity of unfractionated T lymphocytes and their subpopulations bearing Fc-receptors for IgM (TM) and for IgG (TG) was measured. The unfractionated T cells and the TM fraction showed helper activity, whereas the TG cells expressed suppressor activity. The TL-CFC grown in agar in the presence of PHA manifested helper activity at low cell concentration. However, increasing the TL-CFC concentration finally caused suppression of B cell differentiation. The suppressor effect could be abolished by prior irradiation of the TL-CFC before seeding them in agar.