Interleukin-8 as an autocrine growth factor for human colon carcinoma cells in vitro.

Cell lines derived from human colon carcinomas secrete interleukin 8 (IL-8) in vitro and this chemokine has also been detected immunohistochemically in human colon carcinoma specimens, in which it is tumour cell associated. In these experiments, IL-8 was shown to comprise an important component of the angiogenic activity of colon carcinoma cell line supernatants. The effect of modulating IL-8 activity upon the growth of the colon carcinoma cell lines HCT116A, HT29 and CaCo2 was investigated. Supplementing endogenously produced IL-8 by recombinant chemokine led to stimulation of cell growth. Neutralization of the effect of endogenously produced IL-8, either with the specific antagonist peptide AcRRWWCR or with blocking anti-IL-8 antibody, resulted in around 50% inhibition of cell growth (P<0.05). All of the colon carcinoma cell lines tested expressed mRNA for both IL-8RA and RB when grown at confluence. At the protein level, all cell lines expressed IL-8RA. Expression of IL-8RB was weak, although increased expression was seen in HCT116A cells as they approached confluence. Antibodies to IL-8RA and RB did not affect proliferation at low cell density but were strongly inhibitory when cells were cultured at a higher density. These data suggest that IL-8 acts as an autocrine growth factor for colon carcinoma cell lines and would support the concept that a similar autocrine loop operates in vivo.

Combined suicide and granulocyte-macrophage colony-stimulating factor gene therapy induces complete tumor regression and generates antitumor immunity.

The use of prodrug-activated ("suicide") gene therapy has been shown to be effective in inducing tumor regression when only a small proportion of tumor cells contains the suicide gene. These experiments were designed to test whether additional therapeutic benefit may be obtained by stimulating the immune response. Murine MC26 colon carcinoma cells, either untransduced or transduced with genes for herpes simplex virus-1 thymidine kinase (HSV1-TK) or human GM-CSF, were injected subcutaneously into syngeneic BALB/c mice in various combinations. Inoculation of equal numbers of untransduced and HSV1-TK-containing cells followed by ganciclovir (GCV) treatment resulted in almost complete tumor regression, but by 7 weeks, tumors had recurred in all mice. A similar initial regression was obtained using equal numbers of cells containing HSV1-TK and GM-CSF genes, but >80% of these mice remained tumor-free after 3 months. Groups of tumor-free mice that had received GM-CSF-containing cells were left for different periods of time and rechallenged with unmodified MC26 cells on the opposite flank. Of the mice rechallenged 14, 28, and 108 days later, 100%, 88%, and 57%, respectively, showed complete resistance to unmodified tumor cells. In mice that showed tumor regrowth, tumor volume was much less than in control mice. Adoptive transfer of spleen cells from resistant mice to naïve syngeneic mice resulted in partial resistance to challenge with unmodified tumor cells. Specific cytotoxicity against MC26 cells was only demonstrable in mice receiving GM-CSF- and HSV1-TK-containing tumor cells. These experiments show that the presence of cells secreting GM-CSF in HSV1-TK-containing, regressing tumor is able to induce complete or partial resistance to tumor rechallenge. This indicates the potential usefulness of GM-CSF in enhancing other antitumor therapies.

beta-catenin expression in primary and metastatic colorectal carcinoma.

beta-catenin plays a fundamental role in the regulation of the E-cadherin-catenin cell adhesion complex. It also functions in growth signalling events, independently of the cadherin-catenin complex, and these signalling pathways are disturbed in colorectal cancer. Mutations in either the APC or beta-catenin genes in colorectal cancer cells result in up-regulation of protein expression and subsequent cytoplasmic and nuclear distribution of beta-catenin. In this study, we examined beta-catenin expression in 47 primary colorectal tumors and the corresponding liver metastases. Immunohistochemical studies demonstrated loss of membranous beta-catenin expression in 26% of primary tumors and 60% of liver metastases and a concomitant increase in cytoplasmic and nuclear staining. Widespread nuclear expression of beta-catenin was found in 64% of primary tumors and 21% of liver metastases. No associations were found between any form of beta-catenin expression and either tumor stage or tumor grade. Cellular distribution of beta-catenin was also examined by detergent extraction and Western blot analysis in 16 primary tumors and 23 liver metastases. This analysis showed that most tumors demonstrated reduced beta-catenin in the cytoskeletal fraction and increased beta-catenin in the cytosolic fraction. Furthermore, 3 liver metastases were found to contain a truncated beta-catenin protein of approximately M(r) 80,000. Immunoprecipitation studies showed that the truncated beta-catenin proteins only bound weakly to E-cadherin and beta-catenin compared with non-truncated beta-catenin. These results demonstrate gross alterations in the cellular distribution of beta-catenin in primary colorectal cancers with metastatic potential, as well as in the metastatic tumors. These changes may be the consequence of APC or beta-catenin gene mutations, or possibly result from a post-translational modification of the E-cadherin-catenin complex.

Cadherin-catenin expression in primary colorectal cancer: a survival analysis.

Both cell adhesion and cell signalling events are mediated by components of the cadherin-catenin complex. Loss of expression of the components of this complex have been shown to correlate with invasive behaviour in many tumour types although their exact role in colorectal cancer remains unclear. Immunohistochemical analysis of the expression of components of the cadherin-catenin complex in colorectal cancers from 60 patients was undertaken. Loss of memberanous expression of E-cadherin, alpha-catenin and beta-catenin was demonstrated in 52%, 85% and 40% of tumours respectively. Focal nuclear expression of beta-catenin (< 75% of cells per section), usually associated with cytoplasmic expression, was clearly demonstrated in 19 (32%) tumours while widespread nuclear expression (> 75% of tumour cells per section) was seen in 11 (18%) tumours. Loss of membranous alpha-catenin expression significantly correlated with tumour de-differentiation (P = 0.009). There was a trend towards an association between advanced tumour stage and loss of membranous expression of alpha-catenin or beta-catenin, although these associations were not statistically significant. Univariate analysis revealed that advanced Dukes' stage, tumour de-differentiation, loss of membranous beta-catenin expression, cytoplasmic beta-catenin expression and widespread nuclear expression of beta-catenin all correlated with short survival following apparently curative resection of the primary tumour. However, only Dukes' stage (P = 0.002), tumour grade (P = 0.02) and widespread nuclear expression of beta-catenin (P = 0.002) were independent predictors of short survival. Disturbed growth signalling events in colorectal tumours are thought to result in nuclear accumulation of beta-catenin. Consequently, tumours with widespread nuclear expression of beta-catenin are likely to have severely abnormal growth characteristics, and which therefore might be predictive of short survival in these patients.

p53 protein expression in tumours from head and neck subsites, larynx and hypopharynx, and differences in relationship to survival.

The present study involves an immunohistochemical analysis of p53 protein expression in head and neck tumours located at two separate subsites, the larynx and hypopharynx. It attempts to relate differences in expression to differences in the behaviour of these tumours. Detection of the p53 protein was performed using immunohistochemistry on 32 specimens of hypopharyngeal squamous cell carcinoma and 35 specimens of laryngeal squamous cell carcinoma. p53 overexpression was found in 66% of the hypopharyngeal tumours and in 51% of the laryngeal specimens analysed. Some differences between the two tumour types were noted in the pattern staining. p53 staining in those with hypopharyngeal tumours was associated with a statistically significant increased survival. For laryngeal carcinoma the converse was true but did not reach statistical significance. Differences in the behaviour of different head and neck tumour types may be reflected in differences in expression of the p53 protein. While p53 protein expression does not appear to be a useful prognostic indicator in laryngeal carcinoma it might be a useful prognostic indicator in tumours of the hypopharynx. Moreover, it may help predict those tumours which are radioresistant, thus suggesting other modes of treatment for these tumours. Of particular importance is the molecular basis for the observed differences in survival associated with p53 expression in the two tumour sites. This is under further investigation.

Tumor resistance to antimetabolites.

1. The inherent or acquired resistance of certain tumors to cytotoxic drug therapy is a major clinical problem. 2. Resistance to the chemotherapeutic antimetabolites (e.g., methotrexate, 5-fluorouracil, tomudex and gemcitabine) is no exception. 3. Mechanisms of resistance include mutations, amplification of target genes, altered drug transport, differences in nucleoside and nucleobase salvage pathways, DNA damage-response pathways and cell cycle control pathways.

Invasion of human colorectal carcinoma cells is promoted by endogenous basic fibroblast growth factor.

The growth-stimulatory and invasion-promoting effects of basic fibroblast growth factor (bFGF) were examined in 2 series of related human colon carcinoma cell lines (HCT116A, HCT116B and 20-10-1 as well as and LS180, LS174T and ARK1A) that exhibit different invasive potentials. The invasive cell lines 20-10-1 and ARK1A grew more rapidly than their non-invasive counterparts; exogenously added bFGF stimulated the proliferation of all the cells. When extracts of the cells were fractionated on columns of heparin-Sepharose, bFGF-like activity was found in extracts from each cell line. The amount of bFGF-like growth-stimulatory activity was greater in the more invasive cells: the invasive cells 20-10-1 contained 35-fold more activity than the non-invasive HCT116A cells, and the ARK1A cells contained 15-fold more activity than LS180 cells. Relatively small amounts of bFGF-like activity were recovered from medium conditioned by the invasive cells. The bFGF-like growth-stimulatory activity from the cell extracts was neutralised by an antibody to bFGF, and immunoblotting revealed the presence of an 18 kDa immunoreactive polypeptide, consistent with the presence of bFGF in the cell extracts. Exogenously added bFGF caused the usually non-invasive HCT116A cells to invade collagen gels. The HCT116B and 20-10-1 cells that were naturally invasive in a collagen gel assay also showed increased levels of invasiveness in the presence of bFGF, but an antibody that neutralised the activity of bFGF reduced the constitutive invasiveness of these cells. Our results suggest a causal relationship between the endogenous production of bFGF and the invasive potential of human colon carcinoma cells.

Resistance to chemotherapeutic antimetabolites: a function of salvage pathway involvement and cellular response to DNA damage.

The inherent or acquired (induced) resistance of certain tumours to cytotoxic drug therapy is a major clinical problem. There are many categories of cytotoxic agent: the antimetabolites, e.g. methotrexate (MTX), N-phosphonacetyl-L-aspartate (PALA), 5-fluorouracil (5-FU), 6-mercaptopurine (6-TG), hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (AraC); the alkylating agents, e.g. the nitrogen mustards and nitrosoureas; the antibiotics, e.g. doxorubicin and mitomycin C; the plant alkaloids, e.g. vincristine and vinblastine; and miscellaneous compounds, such as cisplatin. There are also many mechanisms of drug resistance elucidated principally from in vitro studies. These include mutation of target genes, amplification of target and mutated genes, differences in repair capacity, altered drug transport and differences in nucleoside and nucleobase salvage pathways (Fox et al, 1991). The aim of the present review is to evaluate in detail the mechanisms of response of both normal and tumour cells to three chemotherapeutic antimetabolites, MTX, PALA and 5-FU, which are routinely used in the clinic either alone or in combination to treat some of the commonest solid tumours, e.g. breast, colon, gastric and head and neck. The normal and tumour cell response to these agents will be discussed in relation to the operation of the known alternative 'salvage pathways' of DNA synthesis and current theories of DNA damage response.

Expression of the E-cadherin-catenin cell adhesion complex in primary squamous cell carcinomas of the head and neck and their nodal metastases.

Reductions in cell-cell adhesion and stromal and vascular invasion are essential steps in the progression from localized malignancy to metastatic disease. In this study, changes in the expression of the components of the E-cadherin-catenin cell adhesion complex have been investigated using immunohistochemical techniques in primary tumours and nodal metastases from 36 patients with squamous cell carcinoma of the head and neck. For 14 patients the corresponding primary and nodal metastases samples were available. None of the 51 samples showed normal E-cadherin expression when compared with either the adjacent normal squamous epithelium or with normal colonic epithelium that was used as positive control material. In 88% of primary tumours fewer than 50% of cells exhibited normal membranous E-cadherin expression. Loss of membranous E-cadherin expression was more extensive in poorly differentiated carcinomas while, in individual carcinomas, membranous E-cadherin expression was stronger in those parts of the neoplasm that expressed the differentiation marker involucrin. Expression of beta-catenin generally paralleled that of E-cadherin, but in 12 cases there was strong membranous beta-catenin expression in samples that exhibited predominantly cytoplasmic E-cadherin labelling. Expression of alpha-catenin was generally weak and did not correlate with the expression of either beta-catenin or E-cadherin. Marked intratumoral heterogeneity for protein expression was evident for all antibodies, and the abnormal expression of the catenins is a novel finding. E-cadherin is expressed more intensely in cells with greater squamous differentiation, but there was no correlation between the decreased expression of any of the adhesion molecules of the E-cadherin complex tested and local recurrence, metastasis or survival. The loss of expression of components of the E-cadherin complex is a common abnormality in squamous carcinomas and, while it may be permissive for metastasis, it does not appear to be the only determinant of this process.

Management strategies for colorectal liver metastases--Part I.

Colorectal cancer is a common malignancy and the incidence of this disease is increasing. Approximately 50% of patients with colorectal cancer die from recurrent disease following an apparently curative resection of the primary tumour and the liver is the most frequent site of relapse. Although only a small proportion of patients will benefit from resection of liver metastases, this form of treatment offers the only possibility of cure. In selected patients, 5-year survival rates of 25-35% may be achieved following liver resection. A poor prognosis after resection of hepatic metastases is likely when there are more than three metastatic deposits, involved resection margins often as a result of ¿wedge' resections, when there is extrahepatic disease, or when there is nodal involvement at the primary tumour site. Regional hepatic artery infusion chemotherapy may provide palliation and possibly even prolongation of survival for some patients with unresectable metastases. Cytoreductive techniques may also provide palliation in selected patients with hepatic metastases unsuitable for resection; cryotherapy is the most promising of these methods.

Management strategies for colorectal liver metastases--Part II.

Liver metastases are relatively common in colorectal cancer and a small proportion of patients may benefit from resection of these liver metastases. In a selected subgroup of patients, 5-year survival rates of 25-35% may be achieved following liver resection. These survival figures compare favourably with those of patients with untreated liver secondaries. In the second part of this review the surgical and non-surgical treatment options for treating colorectal liver metastases are examined in detail.

Fibroblast growth factors and their receptors.

Fibroblast growth factors (FGFs) represent a group of polypeptide mitogens eliciting a wide variety of responses depending upon the target cell type. The knowledge of the cell surface receptors mediating the effects of FGFs has recently expanded remarkably. The complexity of the FGF family and the FGF-induced responses is reflected in the diversity and redundancy of the FGF receptors. In this review, a number of biochemical characteristics and biological properties of the FGF family and its receptors are described and their expression both in normal tissues and in tumours is discussed. Finally we speculate on the targetting of growth inhibition agents to tumours through FGF receptors.

Prodrug-activated gene therapy: involvement of an immunological component in the "bystander effect".

The integration and expression of the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene in localized tumors results in tumor regression after the administration of the specific nucleoside analogue ganciclovir (GCV). Although only 10% to 20% of the tumor cells take up the HSV1-TK gene, the neighboring cells also die, a phenomenon termed "bystander effect.". In the present study, coinjection of the MC26 mouse colon carcinoma cell line and the HSV1-TK expressing retroviral packaging cell line followed after 7 days by the intraperitoneal administration of GCV resulted in almost total tumor regression in the immunocompetent BALB/c mice but not in immunocompromised athymic BALB/c mice. This suggested a strong cell-mediated immune component to the bystander effect.

Differences in resistance to 5-fluorouracil as a function of cell cycle delay and not apoptosis.

A series of human embryo fibroblasts has previously been shown to display increasing resistance to the antimetabolites methotrexate (MTX) and N-phosphonacetyl-L-aspartate (PALA) with increasing tumorigenicity. This increased resistance was found to be further increased as a result of salvage pathway activity for purine and pyrimidine biosynthesis. A similar pattern of increasing resistance paralleling increasing tumorigenicity has now been shown to occur with 5-fluorouracil (5-FU), which is independent of salvage pathway activity. The KMS normal cell line was found to be more sensitive to 5-FU than either the immortalised KMST or tumorigenic KN-NM cell lines. Immunohistochemical analysis of the three cell lines demonstrated high levels of p53 protein in the KMST and KN-NM cell lines, but undetectable p53 levels in the KMS cell line. From these data it was hypothesised that a difference in p53 function may be causing the difference in the patterns of sensitivity observed in the three cell lines. P53 is now believed to function as a regulator of a G1 to S cell cycle checkpoint and as an inducer of apoptosis following DNA damage to the cell. The differences in sensitivity of the cell lines could not be explained by differences in the levels of apoptosis but could be attributed to differences in cell cycle response. Our evidence suggests that loss of cell cycle control, possibly through loss of p53 function, is an important factor in increasing the drug resistance of fibroblast cell lines.

Gene therapy in surgical oncology.

BACKGROUND: Surgery remains the only potentially curative treatment modality for the majority of patients with solid tumors. Conventional chemotherapy and radiotherapy only have roles as adjuvant or palliative therapies for most common cancers. Two decades of research have led to the first attempts at producing and introducing clinically useful genetically modified cells into humans. METHODS: Modern molecular methods have been developed that allow the stable transfer of foreign DNA sequences into human and other mammalian somatic cells. At the present time, gene therapy predominantly involves gene insertion either directly into a target cell in situ or into an appropriate cell in vitro that is then introduced to a physiologically relevant site. We present an overview of the potential applications of molecular biology for practicing surgeons, particularly in the field of surgical oncology, to show how genes are harnessed and inserted into target somatic cells. CONCLUSIONS: Although significant clinical therapies have and will continue to emerge from these initial experiments, only the future will provide evidence of whether the present technical skills are sufficient to have an impact on the long-term benefits for patients with cancer and genetic defects.

Expression of the cell-cell adhesion molecule E-cadherin in tongue carcinoma cell lines.

A reduction in cell adhesiveness and cell invasion are essential steps in tumour progression. In the present study six tongue carcinoma cell lines were compared with regard to their invasive potential in two in vitro invasion assay systems and for their patterns of expression of the cell-cell adhesion molecule E-cadherin. The three cell lines negative for E-cadherin expression were invasive in both assays. One cell line with strong E-cadherin expression was strongly invasive and one weakly invasive. One cell line with reduced E-cadherin expression was weakly invasive. There was no significant pattern to these findings (chi 2 = 0.375; p = 0.54). This supports previous studies from this group that suggest that E-cadherin is only one of the presumably many molecules involved in tumour progression in squamous cell carcinoma of the tongue.

Reduced E-cadherin expression correlates with increased invasiveness in colorectal carcinoma cell lines.

A reduction in cell adhesiveness and cell invasion are essential steps in tumour progression to metastasis. In the present study two out of seven colorectal carcinoma cell lines exhibited reduced expression of the cell-cell adhesion molecule E-cadherin as assessed by immunofluorescence. The same two cell lines were invasive in the collagen gel and membrane invasion culture system invasion assays. Addition of anti-E-cadherin antibody to a non-invasive carcinoma cell line caused the cells to assume a dissociated morphology on plastic and to become invasive in collagen gels. This demonstrates a causal role for E-cadherin in the maintenance of intercellular adhesion and the suppression of tumour cell invasion and possibly metastasis in colorectal tumour cells.

Expression of the cell-cell adhesion molecule E-cadherin in squamous cell carcinoma of the head and neck.

The expression of the cell-cell adhesion molecule E-cadherin has previously been shown to be reduced in poorly differentiated squamous cell carcinoma of the head and neck and absent in nodal metastases. Twenty-eight patients with previously untreated squamous cell carcinoma of the head and neck, 22 of whom had nodal metastases at presentation, were investigated for E-cadherin expression using the monoclonal antibody 6F9, specific for human E-cadherin. Reduced expression was seen in the poorly differentiated primary tumours, compared with well differentiated tumours, but this trend was not statistically significant. E-cadherin expression was present at a reduced level in nodal metastases. It was also noted that, where both the primary tumour and corresponding nodal metastasis were investigated, E-cadherin expression was identical for both samples. The degree of E-cadherin expression did not correlate with survival. These data confirm a reduction in E-cadherin expression in poorly differentiated tumours. There was no correlation between E-cadherin expression and any of the host, tumour and treatment factors associated with malignancies of the head and neck region.