Expression of oncoproteins in primary human non-small cell lung cancer and incidence of metastases

In the current study the relationship between the incidence of metastatic spread and expression (at the protein level) of various proto-oncogenes was investigated in 217 human non-small cell lung carcinomas. Tumors with an overexpression of proteins encoded by the oncogenes c-jun and c-myc showed a significantly increased formation of metastases (c-jun: P = 0.008; c-myc: P = 0.018). No significant correlations were found between the expression of the c-fos, c-erbB1, c-neu and c-ras products and metastatic spread.     

TRANSFORMING GROWTH FACTOR-α EXPRESSION IS ALTERED DURING EXPERIMENTAL HEPATOCARCINOGENESIS

In order to characterize the role of transforming growth factor-α (TGFα) during hepatocarcinogenesis, liver tissue was examined at 10, 16, and 19 weeks following initial 10-week diethylnitrosamine (50 mg l⁻¹ drinking water) exposure in female Wistar rats. Liver tissue protein extracts were electrophoresed and transferred to nitrocellulose filters. Levels of tissue-derived TGFα and epidermal growth factor receptor (EGFr) were assessed using an anti-TGFα monoclonal antibody (Ab-1) and an anti-EGFr polyclonal antibody (AB-4), coupled with scanning densitometric quantification. Immunolocalization of TGFα was performed in Bouin's-fixed, paraffin-embedded liver tissue sections. The distribution and intensity of TGFα immunoreactivity varied according to the degree of dysplasia, severely dysplastic cells being strongly immunoreactive. At week 10, mild hepatocyte dysplasia and perivenular inflammation were evident, together with a corresponding increase in perivenular TGFα immunoreactivity. By week 16, foci of moderate to severe dysplasia were observed; at this stage, there was a decrease in perivenular immunoreactivity but a further increase in overall liver tissue TGFα levels. Some ‘altered foci’ and dysplastic nodules showed intense immunoreactivity for TGFα. At these time points, immunodetectable liver EGFr was found to decrease significantly in comparison with normal control tissue. TGFα immunoreactivity was observed in fully developed carcinomas at week 19, although some tumours were negative by immunohistochemistry. The up-regulation of immunodetectable TGFα and the concomitant down-regulation of EGFr demonstrated positive ( P <0·01) and negative ( P <0·001) correlations, respectively, with hepatocyte proliferation indices. These findings suggest that the TGFα/EGFr ligand receptor system may be important during tumour promotion and in the stimulation of continued proliferation in hepatocellular carcinomas.     

Tumour Regression Induced by Co‐administration of MIP‐3α and CpG in an Experimental Model of Colon Carcinoma

CCL20/macrophage inflammatory protein‐3α (MIP‐3α) represents one of the potent chemoattractive proteins for dendritic cells (DCs). Herein, we investigated whether in vivo genetic modification of tumour cells aimed at intratumoural production of MIP‐3α might lead to accumulation of DCs in tumour tissue. Mice injected with CT26, received recombinant adenovirus (Ad) vectors (AdMIP‐3α) expressing MIP‐3α protein. This was complemented by injections of CpG. Interestingly, MIP‐3α gene therapy combined with CpG injections resulted in specific cytotoxicity. This was associated with significant suppression of tumour growth rate. These findings demonstrate the potential of strategies that utilize in vivo overexpression of chemokines.     

Stem cells in breast epithelia

The rodent and human nonpregnant mammary glands contain epithelial, intermediate and myoepithelial cells which have all been isolated as cell lines in vitro. Transforming growth factor-α (TGFα) and basic fibroblast growth factor (bFGF) are produced by myoepithelial cells and can stimulate the growth of intermediate stem cells in vitro. Epithelial and intermediate cells behave like stem cells in vitro, since they can differentiate into alveolar-like and myoepithelial cells. The myoepithelial differentiation pathway is associated with the early expression of a calcium-binding regulatory protein called p9Ka and the protease, Cathepsin D. Myoepithelial cells are also present in benign lesions but not in malignant mammary carcinomas of rats or humans, whose resultant cell lines fail to differentiate completely along the myoepithelial cell pathway. Loss of the myoepithelial cell in some invasive carcinomas may be compensated, at least in part, by changes in malignant cells. Over-expression of TGFα and/or erbB receptors may reduce the requirement for TGFα, whilst ectopic production of bFGF and its receptors and p9Ka/Cathespin D may assist in tumorigenesis and in metastasis, respectively. Thus compensation for, or retention of, molecules potentially involved in the differentiation of mammary cells may be a mechanism by which malignancy progresses in some human invasive carcinomas.     

Immunocytochemical demonstration of p21ras in normal and transitional cell carcinoma urothelium

Activation and/or overexpression of the protein product of the ras gene family (p21^ras) has been implicated in the development of various cancers, including bladder carcinoma. We have used the anti-p21^ras monoclonal antibody, RAP-5, to assess the level and pattern of expression in formalin-fixed, paraffin-embedded tissue sections of both normal and malignant urothelium. All 14 random normal bladder biopsies and 67 of 68 transitional cell carcinomas of the urinary bladder were positively stained with the RAP-5 antibody. In normal urothelium, p21^ras staining tended to be localized to the superficial cell layer. With increasing histological grade and/or depth of invasion of the tumour, a greater proportion of tissue sections demonstrated a staining pattern which was more uniform with respect to the different epithelial cell types. Serially diluting the primary antibody did not reveal any significant differences in the staining patterns observed. Despite the change in staining pattern with increasing grade, these results suggest that p21^ras expression by itself is not a useful indicator of the malignant phenotype.     

Expression of c-myc protein is related to cell proliferation and expression of growth factor receptors in transitional cell bladder cancer

Archival biopsy specimens from transitional cell bladder tumours (n=185) were analysed immunohistochemically for expression of c-myc protein. The results were compared with compared with histopathological and clinical parameters and survival. Forty-three per cent of the tumours were negative for c-myc protein and weak, moderate, or strong cytoplasmic expression was found in 34, 14, and 9 per cent of cases, respectively. Nuclear positivity for c-myc protein was detected in 35 per cent of tumours and nuclear opositivity was related to overexpression of c-erb B-2 (P=0.01) and a high proportion of nuclei were also positive for p53 oncoprotein (p<0.05). Cytoplasmic expression of c-myc protein was related to histological grade (P=0.005), papillary status (P=0.007), the S-phase fraction (P=0.008), the mitotic index (P=0.021), overexpression of epidermal growth factor receptor (P=0.045), and c-erb B-2 (P=0.17). Expression of c-myc protein was not significantly related to the progression of tumours and it had no prognostic value in survival analysis. Independent predictors were the T-category (P<0.001), papillary status. (P=0.001), and S-phase fraction (P=0.061). The results show that while c-myc gene product participates in growth regulation of human bladder cancer cells, it has no independent prognostic significance.     

Epidermal Growth Factor Behaves as a Partial Agonist in Hepatocytes: Effects on DNA Synthesis in Primary Culture and Competition with Transforming Growth Factor α

Abstract

The structurally related mitogens epidermal growth factor (EGF) and transforming growth factor a (TGFα) are believed to exert all their effects via the same receptor. We have compared the effects of EGF and TGFα, and examined their interaction, on DNA synthesis in cultured rat hepatocytes. The potency of the two agents was similar, or slightly higher for EGF, but TGFα stimulated the DNA synthesis more efficiently, producing at high levels a rate of S phase entry that clearly exceeded (two to threefold) that obtained with maximally effective concentrations of EGF. While the hepatocytes became more sensitive both to TGFα and EGF when addition of the agents was postponed until late in the prereplicative period, TGFα exhibited higher efficacy than EGF both at early and late exposure. When EGF and TGFα were added together at 24 h, TGFα further enhanced the DNA synthesis in the presence of a saturating concentration (5 nM) of EGF, while EGF dose-dependently reduced the DNA synthesis in the presence of a high concentration (10 nM) of TGFα. The results show a lower efficacy of EGF than of TGFα, and, therefore, EGF displays the characteristics of a partial agonist in its EGF receptor-mediated growth stimulation in hepatocytes.     

Influence of TNFα and LTα single nucleotide polymorphisms on susceptibility to and prognosis in cutaneous malignant melanoma in the British population

Cutaneous malignant melanoma (CMM) is a potentially fatal malignancy in which exposure to UV light is the most important risk factor. Several lines of evidence suggest that increased expression of tumour necrosis factor (TNF) α, upregulated by UV exposure, may contribute to tumour escape from the immune response. In this study, we addressed whether single nucleotide polymorphisms (SNPs) in the TNFα promoter and lymphotoxin (LT) α gene are associated with susceptibility to or known prognostic indicators (e.g. initial tumour growth phase, Breslow thickness, mitotic count in vertical growth phase tumours, and tumour regression) in CMM. One hundred and forty‐six British Caucasian CMM patients and 220 controls were typed for TNFα?376, ?308 and ?238 and LTα+252 SNPs by ARMS‐PCR. Only the TNFα?238 GG (P = 0.05) and GA (P = 0.03) genotypes showed slight, but significant, associations with CMM, while LTα+252 AA was associated with a higher mitotic count in vertical growth phase tumours (P = 0.02). Both TNFα?238 and LTα+252 SNPs showed linkage disequilibrium with HLA‐DQB1*0303 and *0301 alleles, variably implicated in CMM susceptibility/prognosis. In addition, TNFα?238, ?308, LTα+252 haplotypes were assigned and compared. The GGA haplotype showed a modest association with CMM (P = 0.04) and with stage of disease (P = 0.03) and initial growth phase in CMM (P = 0.02), but these associations were only significant when P‐values were uncorrected. Unlike basal cell carcinoma, these preliminary findings suggest that genetic variation associated with differential TNFα and LTα production is unlikely to play a major, independent role in susceptibility to, and perhaps prognosis in, CMM.     

Expression of Trefoil Peptides in The Gastric Mucosa of Transgenic Mice Overexpressing Transforming Growth Factor-α

Abstract

Overexpression of transforming growth factor-α (TGF-α) in the gastric mucosa of metallothionein-TGFα(MT-TGFα) transgenic mice leads to a marked alteration in the ontogeny of the fundic cellular lineages. Induction of the transgene leads to the over-production of mucous cells with a concomitant diminution in the development of parietal cell and chief cell lineages. We have sought to define more precisely the mucous cell lineages involved in the mucous cell hyperplasia in MT-TGFα mice by investigating the expression of trefoil peptides in MT-TGFα mice. MT-TGFα mice and their non-transgenic littermates were treated with cadmium sulfate beginning at 13 days of age. Animals were then sacrificed at intervals over the following 2 weeks and gastric mucosa was examined for expression of trefoil peptides and TGFα by immunohistochemistry and in situ hybridization. No TGFα mRNA expression could be demonstrated by in situ hybridization in non-transgenic mice. In MT-TGFα mice, in situ grains for TGFα mRNA were detected at the base of fundic glands in 13 day old animals, whereas the expression was observed more widely in the mucosa of older animals (28 days). TGFα immunoreactivity was observed in foveolar mucous cells and residual parietal cells in MT-TGFα mice at all ages. By in situ hybridization, pS2 mRNA was detected in the surface mucous cells in normal gastric mucosa. In MT-TGFα mice, pS2 mRNA was found throughout the expanded foveolar region. By in situ hybridization, spasmolytic peptide (SP) expression was observed in the region of the progenitor zone in both groups of mice. By immunohistochemistry, SP expression was noted in a broad band of mucous neck cells deep to the progenitor zone. No gastric expression of intestinal trefoil factor (ITF) was noted in either group of mice. The results demonstrate that the expansion of the foveolar mucous cell compartment in MT-TGFα mice is due to the hyperplasia of normal surface cells expressing their particular mucin-associated trefoil peptide, pS2.     

Effects of EGF and TGFβ1 on c‐myc gene expression and DNA synthesis in embryonic hamster palate mesenchymal cells

Previous work has shown that cell proliferation is a major contributor to the early palate morphogenesis in mammals. The present study was undertaken to examine the effect of EGF, TGFβ₁ and their combination on proliferation (measured by DNA synthesis) and on the expression of a growth related proto‐oncogene, c‐myc, in embryonic hamster palate mesenchymal cells (HPMC). Vertically developing hamster palatal shelves were dissected on day 11 of gestation, and trypsinized, and primary cultures were grown in DMEM + 10% serum at 37°C and 5% CO₂. Following appropriate growth factor treatment of HPMC, DNA synthesis was measured by scintillation counting and extracted RNA was subjected to Northern blot analysis. In serum‐starved, pre‐confuent cultures treated with EGF (20 ng/ml), DNA synthesis was stimulated in the presence of 2.5% serum. In contrast, treatment of HPMC with TGFβ₁ (10 ng/ml) in the presence or absence of EGF/serum for 24 hr, or HPMC pre‐treatment with TGFβ₁ (30 min) followed by EGF/serum (24 hr), resulted in an arrest of DNA synthesis. Northern blot analysis of RNA extracted from HPMC showed that as serum‐starved, growth‐arrested cells progressed through G0 to G1 phase of the cell cycle, following EGF treatment, c‐myc was expressed by 1 hr and declined thereafter. In contrast, TGFβ₁ did not support expression of c‐myc. Following pre‐ or co‐treatment with TGFβ₁, the EGF ± serum‐induced expression of c‐myc was seen between 1 and 6 hr. It appears that EGF‐induced expression of c‐myc may be involved in advancing the HPMC in G1, and thus may contribute to the onset of DNA synthesis in HPMC. Since co‐ or pre‐treatment with TGFβ₁ did not inhibit EGF/serum induced expression of c‐myc, it is possible that growth arresting effect of TGFβ₁ may not be exerted directly through inhibition or blockage of c‐myc expression. Anat Rec 254:453–464, 1999. © 1999 Wiley‐Liss, Inc.     

Expression of platelet-derived growth factor and c-myc in atherosclerotic lesions in cholesterol-fed chickens: immunohistochemical and in situ hybridization study

Immunohistochemical examination showed no significant expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, PDGF receptors, or of c-myc in the thoracic and abdominal aortas of normal roosters. In cholesterol-fed roosters, intense immunohistochemical reaction for PDGF-B, PDGF receptor, and c-myc was seen in the lipid-rich thickened intimal lesions of the thoracic and abdominal aortas while no significant immunoreaction for PDGF-A was demonstrated in the same lesions. In accordance with immunohistochemical findings, in situ hybridization demonstrated a significant level of expression of PDGF-B, PDGF-A receptor, PDGF-B receptor, and c-myc genes in proliferating intimal cells of the thoracic and abdominal aortas. These results suggest that coordinate actions of PDGF-B and c-myc play an important role in proliferation of intimal cells in the developing atherosclerotic lesions in chickens.     

Preneoplastic potential of morphologically distinct transformed foci induced by 3-methylcholanthrene

Individual variability of scoring foci positive for transformation presents a difficult problem in assessing the transformation assay. In this study, an attempt was made to identify five morphologically distinct types of transformed foci based on size (2–3, 3–4, and ≥4 mm in diameter), invasiveness (smooth vs. invading margins), and other properties (piling vs. spread) induced by 3-methylcholanthrene in Balb/c-3T3 cells. The transformed focal cells were used in in vitro studies including anchorage-independent analysis, focal reconstruction, gene transfection using NIH-3T3 host cells, and Southern blotting to assess amplification of five proto-oncogenes (K-ras, H-ras, c-fos, c-jun, c-myc) and a tumor suppressor (p53) gene. Results showed that 1) there was a significant increase in anchorage-independent growth of all five types of foci ranging from 7–12%; 2) all five morphological types of transformed foci showed 8–15% focal reconstruction; 3) DNA from all five types of transformed foci induced transformation in NIH-3T3 cells at a level significantly above the control DNA; 4) gene amplification studies indicated amplification in both K-ras and H-ras proto-oncogenes; however, c-fos, c-jun, and c-myc did not show DNA amplification. The tumor suppressor gene (p53) was activated and the increase was up to 3-fold over the normal Balb/c-3T3 DNA. These findings are consistent with our hypothesis that all five morphologically different foci have preneoplastic potential and that any foci of size ≥ 2 mm regardless of invasiveness and piling should be scored as positive during the transformation assay. Environ. Mol. Mutagen. 32:369–376, 1998 © 1998 Wiley-Liss, Inc.     

Ras levels and metalloproteinase activity in normal versus neoplastic rat mammary tissues

We have previously reported that activatedras oncogenes can simultaneously switch on the metastatic phenotype and increased capability to degrade type IV collagen [36]. Here the relationship between c-H-ras, metalloproteinase expression and metastatic behavior was studied inN-nitrosomethylurea (NMU)-induced rat mammary carcinomas, which are known to possess activated c-H-ras. When comparing normal rat breast tissue to mammary carcinomas there was no direct relationship betweenras DNA levels and neoplastic changes. Furthermore, there were no consistent differences between metastatic and non-metastatic carcinomas, or between primary tumors and metastases. The NMU-induced rat mammary carcinomas expressed two major gelatinolytic metal-loproteinases (gelatinases) of 65 and 92kD, but only the 65kD gelatinase was detected in normal breast tissue and a rat fibroma. Type IV collagenolytic activity per 5 g of protein was two to three times higher in the mammary carcinomas than in the normal breasts, whereas the primary tumors did not differ from the corresponding metastases. This study shows thatras amplification is not necessary for development of the malignant or metastatic phenotype in the NMU-induced rat mammary carcinoma model. We have also found that induction of p21ras protein synthesis in a v-H-ras transfected NIH/3T3 (433) cell line, containing a glucocorticoid promoter, does not lead to an increase in metastatic capacity.     

ras Transfection and expression does not induce progression from tumorigenicity to metastatic ability in mouse LTA cells

Studies testing the ability of a transfected ras oncogene to confer metastatic properties on non-metastatic cells have yielded conflicting results. Most of these studies have used recipient cells at early stages of progression (primary or immortalized, non-tumorigenic lines). In this study we tested the ability of the T24-H-ras oncogene to induce progression of tumorigenic, non-metastatic, murine LTA cells to a metastatic phenotype. Metastatic ability was assessed in complementary assays in two immune-deficient hosts, nude mice (after s.c. injection) and chick embryos (after i.v. injection), to determine if ras transfection affected metastatic properties in hosts lacking an intact immune system. Even with greatly elevated levels of ras p21 protein, pools of ras-transfected cells as well as individual clonal populations remained non-metastatic in both hosts. Serial in vivo passaging did not consistently enhance for either ras expression or metastatic ability. We conclude that expression of an activated ras oncogene in LTA cells does not induce progression from a tumorigenic to a metastatic phenotype. These results are in marked contrast to those obtained for ras expression in most other cell types. High levels of expression of an activated ras oncogene thus do not always promote progression from tumorigenicity to metastatic ability.     

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-ras ^H, v-ras ^Ki, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-ras ^H + SV40T and v -ras ^H + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.     

Reduction of invasive potential in K-ras-transformed thyroid cells by restoring of TGF-β pathway

Transforming Growth Factor-beta1 (TGF -β1) is a multifunctional cytokine that regulates a number of cellular processes such as cell growth, differentiation, plasticity, cell motility, adhesiveness, embryogenesis, development and apoptosis through binding to TGF-β receptors. We have previously demonstrated that K-ras-transformed rat thyroid cells, K10, are resistant to the growth inhibitory action of TGF-β1, because they show a decreased expression of type II receptor (TβRII). Clones obtained transfecting TβRII, partially revert their malignant phenotype, showing a reduction in the anchorage-dependent and -independent cell growth and a statistically significant decrease in tumourigenicity with respect to the highly malignant parental cells, both in spontaneous and artificial metastases, when transplanted in athymic nude mice. The purpose of the present work is to elucidate the molecular events involved in the modulation of the tumourigenic potential of K-ras-transformed rat thyroid cells overexpressing TβRII. Our data demonstrate that the TβRII overexpressed in K-ras-transformed thyroid cell clones is a functional receptor and is essential to restore in these cells behaviour similar to that of control cells. The TβRII overexpression is responsible for a strong reduction of adhesive and migratory behaviour of highly malignant K-ras-transformed thyroid cells. These results suggest that the restore of a functional TGF-β receptor in these cells may be useful for the limitation of tumour spread and dissemination.     

Prothymosin α1 effects,in vitro,on the antitumor activity and cytokine production of blood monocytes from colorectal tumor patients

Recent animal studies demonstrate that prothymosin α1 (ProTα) enhances the antitumor response by stimulation of mononuclear phagocyte functions. The present study was aimed at characterizing the in vitro effects by ProTα on blood monocytes from human colon cancer patients. Purified peripheral blood monocytes were studied in terms of tumor cytostatic ability and cytokine production after incubation with ProTα or interferon (rIFN-γ) and transforming growth factor-β (TGFβ), used as reference substances. SW620 colon carcinoma cells were used as tumor target cells in growth inhibition experiments. The level of baseline growth inhibitory activity of unstimulated patient's monocytes was significantly lower than that of normal monocytes. The defective antitumor activity of patient monocytes was associated with a higher production of the inhibitory monokines prostaglandin E₂ (PGE₂) and TGFβ. The stimulation of monocytes by ProTα and/or rIFN-γ elevated the average antitumor activity in all donor groups. The ProTα-induced increase was associated with a significantly higher monocytic secretion of IL-1β and TNF-α. Moreover, the concentrations of TGFβ and PGE₂ in the culture supernatants decreased significantly, when patient's monocytes were treated with ProTα and/or rIFN-γ. Additionally, ProTα enhanced the diminished antitumor activity of TGFβ-treated normal monocytes. These results suggest that ProTα selectively regulates distinct functions of blood monocytes, the effect of this cytokine varying with the parameter and donor population examined. These data provide a rational and biological endpoint for further studies with ProTα as an activator of mononuclear function in colon cancer.