Suppression of the proliferation and migration of oncogenicras-dependent cell lines,cultured in a three-dimensional collagen matrix,by flavonoid-structured molecules

The effects of 7-hydroxycoumarin, genistein and quercetin on tworas-oncogene-driven tumour cells (rat breast adenocarcinoma and human bladder carcinoma) were investigated using cellular (proliferation and migration) and molecular targets (p21 ^ras GTPase activity and intracellular amount of p21 ^ras protein). All three compounds inhibited the growth of both cell lines. Genistein was the most effective substance. Further-more, 7-hydroxycoumarin and genistein affected the motile machinery of both cell lines because major fractions of the cells were slowed down or stopped locomotion. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), a well-known tumour promoter, increased the locomotion behaviour of the cells; the time of migration, the velocity and the distance of migration increased under the control of PMA. 7-Hydroxycoumarin decreased the relative amount of intracellular p21 ^ras , and concomitantly a PMA-induced decrease of p21 ^ras , GTPase activity could be partially antagonized by 7-hydroxycoumarin. Because of the low toxicity and the mode of action evaluated, it is likely that the best role for these substances may be adjuvant therapy of some malignancies following surgery. Profiles directed to migration and proliferation inhibition make these drugs exceptional candidates for chemopreventive strategies in tumours diagnosed as having increasedras oncogene levels.Abbreviations PMA phorbol 12-myristate 13-acetate - PMS phenazine methosulphate - RBA rat breast adenocarcinoma - XTT 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide inner salt, sodium salt     

A serum-free medium for culturing lactogen dependent and autonomous NB2 node lymphoma cells

A serum-free, hormone-free medium (SF2) was designed for the Nb2 rat lymphoma bioassay for lactogens as batches of horse serum (HS), which were commonly used, were found to be inconsistent in their suitability and to contain factors modulating the PRL-induced growth response of clone Nb2-11C. In a 3-day incubation with less than 500 pg/ml human GH (hGH), SF2 was better than the traditional medium in supporting Nb2-11C growth, although the comparative efficiency of SF2 decreased at higher hGH levels. Known growth factors (epidermal growth factor, fibroblast growth factor, platelet derived-growth factor, recombinant somatomedin-C, multiplication-stimulating activity) and insulin had no consistent effect on the cell growth in SF2 either in the presence or absence of hGH. Corticosterone (12.4-150 nM) was toxic to the Nb2-11C cells. SF2 could support the growth of Nb2-11C cells for at least 30 passages in the presence of 5 ng/ml hGH, and that of 2 spontaneously proliferating cell lines (Nb2-SP and Nb2-HSP) for the same length of time in the absence of lactogen. However, in all cases the growth rate in SF2 was lower than that seen in the presence of 10% HS. Long-term culture of Nb2-SP and Nb2-HSP cells in SF2 led to an increase of the growth rate with time. There was a change in the responsiveness of Nb2-SP cells to lactogens after long-term culture in SF2 which was only apparent in the presence of HS. After 10 passages in SF2, Nb2-11C cells showed no apparent changes in lactogen-induced growth response, cell phenotype, cell size, or binding capacity for [125I]hGH.     

Indication of different lactogen and somatogen binding sites in the human growth hormone molecule as probed with monoclonal antibodies

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction has been investigated using as probes monoclonal antibodies (Mabs) to hGH of defined epitope specificity profile. Seven high affinity Mabs were studied for their ability (i) to inhibit the binding of 125I-hGH to Nb2-SP rat lymphoma cells and to IM-9 human lymphocytes possessing lactogen and somatogen type receptors, respectively; and (ii) to interfere with the hormone (hGH or Met8Leu hGH)-induced proliferation in Nb2-11C lymphoma cells. The ability of these Mabs to inhibit the 125I-hGH binding and the hormone-induced proliferation in Nb2-11C cells was negatively correlated with the ability of these Mabs to cross-react with met14 hGH. Furthermore, Mabs Nos. 3 and 7, which cross-reacted minimally (0.2-0.4%) with Met8Leu hGH, were unable to interfere with the mitogenic activity of Met8Leu hGH in Nb2-11C cells. These results indicate that the first 13 amino acids of the N-terminal region of hGH are necessary for its lactogen activity. The inhibition of 125I-hGH binding to IM-9 cells by these Mabs was similar to those observed in Nb2-SP cells, except for Mabs Nos. 19 and 1. These Mabs inhibited more strongly the binding of 125I-hGH to IM-9 than to Nb2 cells and recognized antigenic epitopes close to the C-terminal part of the molecule. These results suggest that the somatogen receptor binding site of hGH may be located on two sites, one at the N-terminal and the other one close to the C-terminal, while the lactogen receptor is mainly confined to the N-terminal part.     

Enhancement of human growth hormone-stimulated mitogenesis of Nb2 node lymphoma cells by 12-O-tetradecanoyl-phorbol-13-acetate

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) enhanced human (h) GH- and ovine PRL-stimulated mitogenesis of the Nb2-11C clone of rat lymphoma cells. Maximal enhancement of 25% in the proliferation rate was achieved with 20 nM TPA. The enhancing effect was found at all levels of hGH (0.031-2.0 ng/ml), but was more pronounced at lower hormone concentrations. TPA alone had no effect on cell proliferation, and its activity was absolutely dependent on the simultaneous presence of the lactogenic hormones. We have analyzed the changes that occurred in the distribution of cells in different phases of the cell cycle during the first 22 h after exposure to hGH and measured the proliferation rate through 3 days. We have found that the mitogenic effect of hGH resulted from 1) an increase in the rate of G0/G1----S transition, 2) a decrease in the lag period required for entry into the S phase, and 3) an increase in the number of cells entering this transition. TPA enhanced all three effects. Binding of [125I]hGH was not affected by prior exposure to TPA, suggesting that the effect of TPA is at a postreceptor level. Proliferation of an autonomous clone of Nb2 cells that does not require lactogenic hormones for growth was not stimulated by TPA, although these cells bound [3H]TPA to the same extent as the PRL-dependent Nb2-11C clone.     

Comparative study of in vitro and in vivo modulation of lactogenic and somatotropic receptors by native human growth hormone and its modified analog prepared by recombinant deoxyribonucleic acid technology

A modified analog of human GH (hGH), prepared by recombinant DNA technology, that lacks 13 amino acids at the amino terminus (Met14hGH), was able to compete with [125I]hGH for binding to lactogenic receptors in Nb2-11C rat lymphoma cells, to somatotropic receptors in IM-9 human lymphocytes, and to both lactogenic and somatotropic receptors in the microsomal fraction of virgin female rat liver. Exposure of intact Nb2 or IM-9 cells to Met14hGH did not reduce the number of surface or intracellular receptors, as compared to the control without hormone. A parallel exposure to 500-fold lower concentrations of hGH resulted in 77-93% reduction in both surface and intracellular receptors. In contrast to [125I]hGH, [125I]Met14hGH was not taken up by the intact Nb2 lymphoma cells. Infusion of anesthetized female virgin rats for 3 h with hGH down-regulated both lactogenic and somatotropic receptors in the liver. A similar infusion with up to 200-fold higher amounts of Met14hGH did not lower the number of total receptors, indicating lack of down-regulation. Some decrease in the binding to free receptors was observed, suggesting that Met14hGH is capable of binding to liver receptors in vivo.     

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3′-kinase in NIH 3T3 cells

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21^ras) and phosphatidylinositol kinase. p21^ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21^ras-bound GTP to p21^ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21^ras. Quiescent cells had dramatically reduced levels of activated p21^ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3′-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.     

Proliferative heterogeneity of human renal cell carcinomas and prevalence ofras gene point mutations

The variable prevalence and a possible stage-dependent increase ofras gene point mutations in human tumors might correspond to clonal growth advantages ofras-activated cells. Tumor areas with activatedras genes might thus differ in proliferative activity from those lackingras gene activation. This hypothesis is studied in a series of human renal cell carcinomas that had been used previously for an analysis of proliferative compartments after post-operative vascular [³H]/[¹⁴C]thymidine perfusion [Rabes et al. (1979) Cancer 44: 799–813]. The growth fraction of different subcompartments of these tumors was studied by immunohistochemistry with mib1 antibody, recognizing a fixation- and embedding-resistant epitope of Ki-67 protein. Thirty subpopulations of 14 human renal cell carcinomas that exhibited a broad spectrum of proliferative activity were chosen for an analysis of the prevalence of K-ras point mutations in exon 1 by a mutation-enriching primermediated restriction-fragment-length-polymorphism analysis and/or direct sequencing of polymerase-chain-reaction-amplified material. The combined autoradiographic and immunohistochemical analysis confirmed the intra- and intertumoral proliferative heterogeneity. Compared to [³H]/[¹⁴C]thymidine labeling indices, mib1 labeling indices are higher. The ratio of mib1 to [³H]/[¹⁴C]thymidine labeling indices varies from 1.9 to 4.1 for the individual tumor subcompartments. However, neither in K-ras codons 12/13 nor in adjacent codons did we detect any mutations in the various tumor compartments. The results suggest that neither mode of proliferation nor type of differentiation is related to K-ras exon 1 point mutations in human renal cell carcinomas.Abbreviations PCR polymerase chain reaction - RCC renal cell carcinoma - BrdUrd 5-bromo-2-deoxyuridine - [³H]dThd [³H]thymidine Supported by a grant from Dr. Mildred Scheel-Stiftung für Krebsforschung, Bonn, F. R. Germany     

Role of H-ras in the malignant progression of rat tracheal epithelial cells

The effect of an activated H-ras oncogene on the progression of neoplasia was studied in transformed rat tracheal epithelial cells. Nude mouse tumours produced by injection of these cells exhibited a higher fraction of cells containing the mutantras gene than did the injected cells, while a subclone that lacked theras mutation was much less tumorigenic than parental cells. Serial passage of one cell line containing aras mutation resulted in an increase in the fraction ofras-mutated cells, which suggests that, in this line,ras activation may confer a selective advantagein vitro as well. However, this was not seen in anotherras-containing line, suggesting the importance of alternative pathways in malignant progression of rat tracheal epithelial cells.Abbreviations RTE rat tracheal epithelial - RFLP restriction-fragment-length polymorphism - DMBA 7,12-dimethylbenz[a]anthracene Supported by NIH grants CA 52925, CA 13343, and ES 00260 and ACS Award IRG-14-33.     

Epithelial Proliferation and ras p21 Oncoprotein Expression in Rectal Mucosa of Patients with Ulcerative Colitis

In ulcerative colitis (UC), epithelial proliferation plays a role in crypt repair and neoplastic evolution. Proliferative status is predominantly connoted in active disease, but not defined in remission. Histologically, remission is characterized by normalization of the picture or development of atrophy. Mutation of the ras oncogene is involved in intestinal carcinogenesis. Aim of this work was to assess the proliferative pattern of rectal epithelium in UC during disease activity and in remission and correlate it with ras oncoprotein p21. The study was performed retrospectively in rectal biopsies from four groups each of 10 patients: active ulcerative colitis (AUC), remission with a normal histology (RUC), remission with rectal atrophy (ARUC), and irritable bowel syndrome (C, control group). In all, immunohistostain was employed to evaluate the proliferation cell nuclear antigen labeling index (PCNA LI) and ras p21. Statistical analysis was performed by ANOVA and Student-Neumann-Keuls tests. %PCNA LI was significantly higher in AUC and ARUC than in RUC and C. Positive cells were predominant in the lower zone of crypts in RUC and C, while a significant expression of PCNA was also observed in the upper areas in AUC and ARUC. Oncoprotein p21 was expressed on the apical surface of the epithelium in 3/10 AUC patients, in all 10 ARUC patients and in none of RUC and C. %The persistently increased epithelial proliferation associated with ras p21 expression in ARUC may be due to the action of an abnormal, mutated ras gene that could play a role in UC-related tumorigenesis.     

Apoptosis of human BEL-7402 hepatocellular carcinoma cells released by antisense H-ras DNA-in vitro and in vivo studies

Recent findings suggest that over-expression of activated H-ras inhibited apoptotic cell death by blocking the activity of apoptotic endonuclease(s). This study was designed using antisense H-ras oligodeoxynucleotides (ODN) to evaluate whether alterations of H-ras expression in BEL-7402 human hepatocellular carcinoma cells could influence the induction of apoptosis in vitro and in vivo. We found that, in vitro, continuous suppression of H-ras expression could decrease the proliferation of BEL-7402 cells and inhibit H-ras-induced entry into S phase. In situ end labeling showed that a large number of cells underwent apoptotic cell death after treatment with antisense H-ras ODN (P<0.01), and gel electrophoresis of DNA extracted from these cells demonstrated a typical DNA ladder, characteristic of apoptosis. In vivo study indicated that pretreatment with antisense H-ras significantly retarded tumor growth in comparison with the untreated controls or tumors treated with non-specific ODN (P<0.01,P<0.01). In situ end-labeling revealed that pronounced apoptotic nuclei were also present in the tissue treated with antisense H-ras ODN (P<0.01). Immunocyto-histochemical study showed that expression of p21^H-ras was significantly decreased after treatment with antisense H-ras. These results indicate that suppression of H-ras over-expression by antisense ODN could effectively inhibit tumor growth and revive the apoptotic pathway by releasing the activity of apoptotic endonuclease(s). The data also suggest the need for further studies to elucidate molecular events involved in antisense H-ras-released apoptosis and evaluate its therapeutic implications.Abbreviations ODN oligodeoxynucleotides - HCC hepatocellular carcinoma - PBS phosphate-buffered saline     

Immunocytochemical detection of p21 ras expression in fresh human leukaemic cells and cell lines

Summary The expression of p21 ^ras proteins was investigated by immunocytochemistry in permanent cell lines and in fresh human leukaemic cells. While high and low levels of p21 ^ras could be detected in most of the cell lines, no significant p21 ^ras immunoreactivity was noted in cells of ten human acute and chronic leukaemias. Thus, notwithstanding its possible role in the initial transformation process in human leukaemias, p21 ^ras expression appears not to be an irrevocable requirement for the maintenance of the transformed state.     

Gene analysis of K-, H-ras,p53, and retinoblastoma susceptibility genes in human lung cancer cell lines by the polymerase chain reaction/single-strand conformation polymorphism method

In order to know the involvement of multiple gene alterations in the pathogenesis of human lung cancer, we examined the genes of K-, H-ras (codons 12, 13, 61), p53(exons 5–9) and the retinoblastoma susceptibility gene (RB)(exons 20–22) using the polymerase chain reaction/single-strand conformation polymorphism method in 32 human lung cancer cell lines (5 squamous-cell carcinomas, 10 adenocarcinomas, 3 large-cell carcinomas, 14 small-cell carcinomas). In 18 non-small-cell lung cancer lines, gene alterations were found in 4 for K-ras (22%), none for H-ras (0%), 4 for p53 (22%) and none for the RB (0%) gene. In 14 small-cell lung cancer (SCLC) lines, no gene alterations were found in K-ras (0%), or H-ras (0%), but 6 were found for p53 (43%) and 3 for the RB (21%) gene. Coincident abnormalities of K-ras and p53, or K-ras and RB genes were not found in any cell lines, and those of the p53 and RB genes were found in only 2 SCLC lines. No association was observed between these three gene alterations and N-myc amplification. Although the above three genes may be involved to some extent in the pathogenesis of lung cancer, more factors are required for its development.Abbreviation PCR-SSCP polymerase chain reaction/single-strand conformation polymorphism     

Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Analysis of Ki-<Emphasis Type="Italic">ras</Emphasis> gene mutations within the same tumor using a single tumor crypt in colorectal carcinomas

Background The point mutations occurring in codons 12 and 13 of the Ki-ras gene are useful genetic markers to identify intratumoral heterogeneity. A single tumor crypt, which consists of monoclonal cells, can be obtained using the crypt isolation method. Ki-ras gene mutations have been examined using the crypt isolation method to determine whether multiclonarity is present within the same tumor.Methods Ki-ras gene mutations were analyzed using a crypt isolation technique coupled with polymerase chain reaction and direct sequencing in 21 sporadic colorectal carcinomas. The specimens were divided into two groups: a representative sample, which was composed of more than 50 tumor crypts, and a single tumor crypt sample. The latter consisted of 10 single tumor crypts, which were obtained from the same tumor separately.Results Ki-ras gene mutations were found in 11 of 21 representative samples and in 12 of 21 single tumor crypt samples. In the 11 samples with Ki-ras mutation, Ki-ras mutations were also found in most single tumor crypts. Among the 12 base substitutions found, G:C to A:T transitions were the most commonly observed. There were no differences between the two samples in the types of Ki-ras mutations found. One Ki-ras mutation that was not detected in the representative sample was observed in only a single tumor crypt.Conclusions Most carcinomas appear to have a homogeneous composition that may result from the successful progression of one of the clones having a Ki-ras mutation. Additional mutations in the Ki-ras gene were rarely observed in colorectal carcinomas.     

Prognostic significance of K-ras andTP53 mutations in the role of adjuvant chemotherapy on survival in patients with dukes C colon cancer

PURPOSE: Mutations in K-ras andTP53 genes are common in colorectal cancer. They affect biologic behavior and might influence chemotherapy susceptibility in these tumors. We investigated whether the survival of patients with Dukes C colon cancer treated with adjuvant chemotherapy is influenced by K-ras andTP53 mutations. METHODS: Mutation screening of the hot spots of the K-ras gene and of the evolutionarily conserved regions of theTP53 gene was performed by denaturing gradient gel electrophoresis technique in formalin-fixed paraffin-embedded specimens of 55 consecutive patients with Dukes C colon cancer treated with adjuvant 5-fluorouracil-based chemotherapy. The median follow-up was 47 (range, 32–66) months. RESULTS: Alterations in the mutation hot spots of K-ras were found at codon 12 (n=11) and 13 (n=4) in 15 of the 55 carcinomas (27 percent). No mutation was found at codon 61. Mutations of a probably causative nature in the evolutionarily conserved regions (exons 5–8) of theTP53 gene were found in 24 tumors (44 percent). K-ras andTP53 mutations were found equally in the group with recurrent disease (7/26 (26 percent) and 12/27 (44 percent), respectively) and in the group without recurrences (8/28 (24 percent) and 12/28 (43 percent), respectively). Cancer-specific survival did not differ significantly between patients with K-ras orTP53 or both mutated and nonmutated tumors, respectively (log-rank test: K-ras, P=0.72 andTP53, P=0.77; K-ras andTP53, P=0.8). Also, potentially aggressive K-ras codon 12 and 13 mutations had the same survival as tumors without these mutations (log-rank test;P=0.73). CONCLUSIONS: Patients with K-ras orTP53 or both mutated Dukes C colon tumors have the same survival as nonmutated tumors when treated with adjuvant chemotherapy. These data suggest that mutations in K-ras orTP53 alone are not prognostic indicators in patients with Dukes C colon cancer receiving adjuvant 5-Fluorouracil-based therapy.Supported by a grant form the Netherlands Cancer Foundation (NKB/KWF).Presented at the European Cancer Conference, Vienna, Austria, Sept 13 to 16, 1999.     

p53 and p14 increase sensitivity of gastric cells to H. pylori-induced apoptosis

H. Pylori infection of the gastric mucosa is associated with increased epithelial cell apoptosis. In vitro, interferon- and TNF- have been shown to increase the sensitivity of cells to apoptosis induced by H. Pylori. The p53 tumor suppressor gene is frequently mutated in many cancers, including gastric cancer. Since p53 protein can induce apoptosis, we sought to determine whether or not p53 increases the ability of gastric epithelial cells to undergo apoptosis in response to H. Pylori-induced cell injury. Human gastric epithelial cell lines, AGS (p53 wild-type) cells and AGS cells infected with HPV E6 gene (AGS-E6) to inactivate p53 were exposed to H. Pylori. The p53, p21, and p14^ARF proteins were measured in gastric epithelial cells by immunoelectrophoresis. Gastric epithelial cell apoptosis was measured by DNA end-labeling assay (TUNEL) and subG₀ cell fractions using flow cytometry, and by agarose gel electrophoresis of DNA. Exposure to H. Pylori increased the levels of p53, p21, and p14^ARF proteins two fold in AGS cells. Gastric AGS cells with fragmented DNA increased from 1.1% to 68% in after exposure to H. Pylori for 24 hr. However, AGS-E6 cells were relatively resistant to apoptosis induced by H. Pylori (only 15% of cells underwent apoptosis). In additional experiments, mouse embryonic fibroblasts (MEFs) were used to further investigate the role of ARF in stabilizing p53 after exposure to H. Pylori. Wild-type and p19^ARF–/– MEFs were exposed to H. Pylori and evaluated for activation of p53, p19^ARF, and apoptosis. As with AGS cells, H. Pylori stimulated a 2-fold increase in p53 and p19^ARF in wild-type MEFs; however, there was no increase in p53 in ARF-null MEFs. H. Pylori easily stimulated apoptosis in wild-type MEFs, although, the absence of p19^ARF significantly reduced the ability of H. Pylori to induce apoptosis in these cells. Activation of ARF by H. Pylori is important in stabilizing p53 resulting in increased apoptosis. Thus, inactivation of either ARF or p53 in gastric cells may reduce their ability to undergo apoptosis in response to injury induced by H. Pylori.     

Production and characterization of monoclonal antibodies toN 7-phenylguanine

N ⁷-Phenylguanine, a base adduct possibly formed after arylation of DNA by benzene oxide, the first reaction metabolite during benzene metabolism, was synthesized in our laboratory and used as reference for the production and characterization of monoclonal antibodies. 2-Hydroxymethyl-7-phenylhypoxanthine, a molecule structurally similar toN ⁷-phenylguanine, was coupled by a linker molecule to different carrier proteins. The resulting conjugate was used to immunize BALB/c mice, the spleen cells of which were fused with mouse P3X63-Ag8.653 myeloma cells to obtain monoclonal antibodies. Several hybridoma lines were cultivated in defined media and characterized as to sensitivity and specificity by an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA demonstrated that all antibodies showed a very high affinity forN ⁷-phenylguanine but had a lower affinity towards various other samples includingN ⁷-chlorophenylguanines andC ⁸-,N ²- andO ⁶-phenylguanine. As little as about 20 pgN ⁷-phenylguanine could be detected with one of the most sensitive antibodies, CE6/G11, with a colorimetric end point while the detection limit could be lowered to about 10 pgN ⁷-phenylguanine when a fluorescent end point was used. The detection limit of other methods used to determineN ⁷-phenylguanine so far is 10 ng for gaschromatography/mass-spectrometry and 1 ng for high-pressure liquid chromatography. Thus the use of specific monoclonal antibodies seems to be the most sensitive method for the detection ofN ⁷-phenylguanine.Abbreviations ABTS 2,2-azino-di(3-ethylbenzthiazolinesulphonate - DMEM Dulbecco's modified Eagle medium - ELISA enzyme-linked immunosorbent assay - HAT hypoxanthine/aminopterin/thymidine - KLH keyhole limpet haemocyanin - PBS phosphate-buffered saline This work was supported by grants from the DLR     

Induction of urokinase activity and malignant phenotype in bladder carcinoma cells after transfection of the activated Ha-ras oncogene

Summary In order to characterize further the previously observed induction of a highly metastatic phenotype in mouse bladder carcinoma cells by Ha-ras transfection, we studied production of plasminogen activator, in vitro invasiveness, and the potential for lung colonization of these cells. The parent carcinoma cells produced predominantly tissue-type plasminogen activator. Out of 13 clones of ras-transfected cells tested, 8 secreted quantitatively elevated levels of plasminogen activator (up to 3.5-fold) as compared to the control transfectants. The plasminogen activator activity in cell lysates was maximally increased 3-fold, the surface-associated activity increased 2.5-fold. The secreted plasminogen activator of cloned ras-transfected cells was characterized to be predominantly of the urokinase type (71.3% compared to 20.5% with the parental BL cells). Thus, in addition to the quantitative augmentation of plasminogen activator production and secretion in a large fraction of the ras-transfected cell population, a significant qualitative shift from tissue-type to urokinase-type has been observed. In addition, ras-transfection augmented the capacity of the cells for invasion into Matrigel in a double-filter in vitro assay as well as their ability to colonize the lungs of syngeneic animals. These malignant properties of the transfected cells might be responsible for their highly metastatic behaviour induced by ras transfection.Abbreviations u-PA urokinase-type plasminogen activator - t-PA tissue-type plasminogen activator - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis     

Carcinogen-induced liver tumours of Wistar rats: absence of activatedras genes and of N-rasC

Summary We examined mutational activation ofras genes in rat liver preneoplasias and tumours induced by diethylnitrosamine andN-methyl-N-nitrosourea (NMU). In accordance with previous reports on H- and K-ras genes, no mutations were detected in the investigated hepatic tumours and prestages suggesting that neither mutations at codons 12, 13 and 61 of H- and N-ras nor a mutation in the last intron of the H-ras gene are involved in initiation and progression of rat hepatocellular carcinomas. In the course of this investigation we found two N-ras genes (N-rasA, N-rasB). Surprisingly N-rasC, which is present in the germ line of Fischer rats, is missing in Wistar rats. This suggests different numbers of germline N-ras genes in members of one species. Two out of eight NMU-induced liver tumours exhibited additional N-ras-related sequences of unknown origin.Abbreviations PCR polymerase chain reaction - NMU N-methyl-N-methyl-N-nitrosourea