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Detection of the c-myc oncogene product in colonic polyps and carcinomas.

The c-myc oncogene has been implicated in the processes of normal cell proliferation and differentiation. Elevated levels of c-myc mRNA and its gene product (p62c-myc), have been detected in a variety of solid tumours and cultured cel lines. Its precise role in normal cell function and in neoplastic transformation and progression has yet to be elucidated. We have used a monoclonal antibody, raised by peptide immunisation, to determine the distribution by immunoperoxidase staining of the c-myc oncogene product in archival specimens of colonic polyps and carcinomas. Samples from 42 patients with colon carcinoma, 24 with benign polyps and 15 normal colon biopsies were examined. Normal colon revealed maximum staining in the mid-zone of the crypts, corresponding to the zone of differentiation and maturation. The staining was predominantly cytoplasmic. Adenomatous polyps revealed the most intense pattern of staining in areas of dysplastic change. Colonic tumours showed a wide range of staining. Well differentiated tumours contained more cytoplasmic p62c-myc than poorly differentiated tumours. These findings suggest that the c-myc oncogene product may play an important role in the evolution of colonic neoplasia. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5

The study of retroviruses has altered our perspectives on the molecular biology of normal and malignant cell proliferation. The discovery of proto-oncogenes, the cellular homologues of viral genes which have transforming ability, has been the basis for this change (Cooper & Lane, 1984). Twenty-five proto-oncogenes have now been identified, molecularly cloned and their sequence determined (Hamlyn & Sikora, 1983). A variety of mechanisms, including gene amplification, translocation and mutation have been identified as abnormal activators of several oncogenes (Burgess, 1985).
Much work has been carried out to identify the functional products of these genes. The c-sis gene has been shown to code for a subunit of platelet derived growth factor (PDGF) (Waterfield et al., 1983) and the c-erb B gene encodes for the intracellular component of the epidermal growth factor (EGF) receptor-suggesting important roles for these oncogene products in growth control (Downward et al., 1984). Abnormalities in the cmyc gene and its expression have been found in a wide variety of tumours. The level of c-myc mRNA increases rapidly when a cell is stimulated to divide and both the mRNA and its gene product, the 62,000 dalton protein (p62c-mYc), both have a short half life of 20 minutes (Rabbitts et al., 1985). Stimulation of fibroblasts by PDGF and EGF results in cell-cycle initiation leading to a subsequent rise in c-myc RNA levels (Kelly et al., 1983). Increased expression of c-myc has been seen in several cell lines when stimulated to proliferate, in regenerating normal liver (Goyette et al., 1983), and during normal embryogenesis (Muller et al., 1982). p62C-MYC is associated with the nuclear fraction of proliferating cells (Eisenman et al., 1985). It also has been found to have DNA binding activity, though Evan and Hancock (1985) have shown that in intact nuclei treated with DNAse at low salt concentration, p62c-mYc is still present. It is probable therefore that the binding of p62c-mYc to DNA is not the only means by which the gene product is associated with the nucleus. These data would suggest that p62c-mYc may have a role in cell cycle control.
It is the breakdown of the normal molecular control mechanisms that result in the neoplastic phenotype. The contribution of abnormal levels of p62c-mYc to the development and maintenance of the malignant state is not currently understood. We have recently developed a set of monoclonal antibodies to p62c-mYc by peptide fragment immunisation (Evan et al., 1985). One such antibody, Myc 1-6E10, recognises p62c-mYc in paraffin wax embedded histological samples (Sikoraet al., 1985). The subcellular localisation of p62c-mYc as detected by this antibody is predominantly cytoplasmic reflecting changes in cellular p62c-mYc distribution due to fixation. Indeed freshly harvested human tumour cell lines stained by immunofluorescence without fixation show clear nuclear fluorescence.
Colorectal carcinoma is a major cause of death from malignant disease. There is good evidence that many large bowel cancers are preceded by a series of adenomatous changes (Mato et al., 1975). Here we report a retrospective immunohistological analysis of colonic specimens using the anti p62c-mYc antibody to obtain an insight into the role of this protein in the adenoma-carcinoma sequence.
Patients and methods Monoclonal antibody The methods employed in peptide synthesis, inoculation of mice and the screening procedures used for deriving Myc-l 6E10 have been fully described elsewhere (Evan et al., 1985). The MCA was raised to a synthetic peptide consisting of residues 171-188 of the human p62c-mYc sequence.
Hybridoma cells were grown in the ascitic fluid of female BALB/c mice. The antibody was purified by octanoic acid precipitation followed by ammonium sulphate concentration. The purified antibody was adjusted to a concentration of 2mgml-' in PBS with 0.001% sodium azide, aliquoted and stored at -20°C. A mouse immunoglobulin of the same subclass (IgG1k) was obtained from the ascitic fluid of the mouse myeloma line X-63 (Kohler & Milstein, 1975). This was similarly purified and also adjusted to a concentration of 2 mg ml1 and was employed as an inappropriate antibody control. Anti-CEA (Dakopat) was employed as a positive control monoclonal antibody.

Immunohistology
Routinely processed formal saline fixed paraffin blocks containing specimens obtained at surgery were extracted from the archives of the Department of Pathology, Addenbrooke's Hospital. Forty-two patients with carcinoma, 24 with benign polyps, and 15 normal colon biopsies were studied. The specimens were sectioned on a bench microtome.
Sections (5 pm) were placed on standard microscope slides which had been previously coated in a 0.5% gelatin solution containing 250 mg chrome alum and 30mg sodium azide per 100 ml and air dried at room temperature overnight.
An avidin-biotin staining technique was employed (Vectorstain, ABC Kit, Vector Labs). Briefly, the sections were dewaxed and rehydrated through alochol. Endogenous peroxidase activity was blocked by incubating for 15min in 5% hydrogen peroxide in PBS (pH 7.2). The sections were washed for 20min in PBS. Excess serum was blotted off and the sections incubated in a moisture chamber for 60 min at room temperature with 100 p1 Myc-1 6E10, diluted 1/500 in PBS, 1% BSA and 0.25% Triton X-100. Sections were washed in PBS and then incubated with a biotinylated rabbit anti-mouse immunoglobulin. After washing, sections were incubated with the ABC reagent. The substrate was developed by a final incubation with a diaminobenzidine solution (20ml PBS, 10mg DAB (Sigma), 20 pl H202 100 vol). The sections were washed in running tap water, counterstained with Mayers haemalum for 60 s, dehydrated, cleared, and mounted.

Results
Specificity controls All carcinoma specimens studied revealed considerable staining using the Myc-I 6E10 antibody. An irrelevant mouse monoclonal immunoglobulin (X-63) of the same Ig subclass revealed no binding. Anti-CEA monoclonal (Dakopats) was used as a positive control. Staining by Myc-I 6E10 but not anti-CEA antibody was blocked by the addition of 1 ug of the peptide used as the immunogen added to 100 M1 of antibody prior to its addition to the section indicating specific antigen recognition (Table I). Normal colon Fifteen samples of normal colon were examined. The presence of p62c-myc in a normal colon biopsy is shown in Figure 1. Maximal staining was present in the transition zone between the actively dividing cells of the crypt base and the mature surface epithelium.
Polyps Samples from 24 patients were studied. Thirteen had villous adenomas and 11 pure adenomas. Ten of these polyps were isolated lesions from patients who had polyps removed endoscopically. The remaining polyps were from patients with co-existing carcinomas. In all polyps p62c-mYc was present in considerable quantities in the crypt cells as well as in the maturing zone (Figures 2 and 3). The staining was mainly cytoplasmic. In some of the larger polyps with dysplastic change the staining was particularly intense and extended to the mucosal surface.
Carcinomas Forty-two patients with carcinomas were investigated. Twenty-nine were classed as moderately to well differentiated and 13 poorly differentiated. The patients were further divided into two groups depending on whether they were alive or dead at five years. The carcinomas showed varying degrees of cytoplasmic staining (Figures 4  and 5). There was no correlation with depth of invasion nor was there any correlation with those patients alive at five years or those dead within five years from their disease. The poorly differentiated tumours showed the least staining. (Summary Table  II).

Discussion
Several oncogenes have been implicated in the development of tumours in patients. Abnormalities in expression of the c-ras gene family have been identified in bladder, colon and lung tumours (Slamon et al., 1984). Abnormal c-myc gene expression has been extensively studied in Burkitt's lymphoma (Melchers et al., 1984). Here chromosomal translocations bring the c-myc gene in close proximity to the immunoglobulin heavy or light chain loci. Other methods of c-myc activation have been discovered in vitro such as the loss of transcriptional repression through deletion of the untranslated 5' exon (Rabbitts et al., 1984) and the abnormal use of promoters in malignant cells (Hamlyn & Rabbitts, 1983). These genetic changes result in an elevated level of either normal or abnormal gene product which subverts the normal growth control pattern resulting in the transformed phenotype. The c-myc gene has been shown to have a role in cell division and differentiation. The analysis of mRNA from human placentae reveals that the peak transcriptional activity occurs between four and five weeks after conception (Pfeifer-Ohlsson et al., 1984). In cells growing in tissue culture the progression from quiescence to active DNA synthesis, the driving of cells from Go into the G1 phase of the cycle, may well be a function of p62c mYc. Several systems have implicated the role of this protein in differentiation (Sikora et al., 1985) although the precise molecular mechanisms involved are unclear.
Staining with Myc-I 6E10 was predominantly cytoplasmic despite the known nuclear location of p62c-mYc from immunochemical studies. The fixation process used for clinical specimens causes the redistribution of p62c-mYc. Fresh human tumour cell lines studied by immunofluorescence show a predominantly nuclear localisation for this protein. This becomes cytoplasmic after fixation (Stewart et al., unpublished). Furthermore, it has been previously demonstrated that p62c-mYc is extracted from nuclei by mild salt concentrations (below 200mM) without affecting gross nuclear structure or causing extraction of major chromatin components. The fractionation of nuclei in the presence of isotonic sodium chloride (144mM) results in virtually quantitative extraction of p62c-myc from nuclei when assayed by immunochemical means (Evan & Hancock, 1985). Despite the lack of information provided on the subcellular localisation  A* of p62c-mYc a quantitative estimate can be obtained of total cellular content using immunohistological techniques. Normal colonic epithelial cells have a rapid turnover rate of 4 to 6 days depending on their type and site. Within the crypts of Lieberkuhn there is considerable variation in the proliferative rate as studied by the uptake of tritiated thymidine (Deschner et al., 1966). This suggests that the cells of the colon are susceptible to different controls or have a range of sensitivities to the molecular triggers for cell cycle entry. Our study suggests that p62c-mYc may be involved in the maturation process for it is present in greatest abundance in the transitional zone of the normal colon ( Figure 6). This has also been observed in the normal colonic mucosa of mice using an MCA to p62c-myc (R. Buick, personal communication). As a result of prolonged exposure to dietary carcinogens and their metabolites genetic changes occur in certain cells resulting in the abnormal accumulation of p62c-myc and subsequent dysplastic change. The lesions most commonly associated with malignant change are those with a villous architecture and which express the greatest quantity of p62c-mYc. Areas of dysplastic change also contain abundant p62c-mYc.
There is evidence that other oncogenes are active in colonic neoplasia, indicating a sequential activation or interplay which would add strength to the argument of multi-stage carcinogenesis (Thor et al., 1984). Gallick et al. (1985) demonstrated elevated levels of p2lras in 9 of 17 primary colonic lesions (8 were graded Dukes stage B or C), however, 4 of 5 tumours classified as having distant metastasis did not show elevated levels. In all the metastases, p2lIas levels were low. They concluded that the elevation of p2lIas may be an early phenomenon in colon cancer and that tumour progression led to an autonomous population of cells in which other growth factors replaced the role of p2lIas. Similar Figure 6 Diagrammatic representation of tritiated thymidine uptake and p62cmYc distribution in normal colon, polyp and carcinoma. Dark shading indicates areas of considerable uptake or staining; unshaded areasno uptake or staining, with stippled areas being in-between (cf. Deschner et al., 1966). examining c-myc mRNA transcripts in polyps and neoplasms (Spandidos et al., 1984).
Elevated p62c-myc expression may therefore either represent the 'driving mechanism' for the increased rate of cell proliferation or it may be a consequence of the activation of other oncogenes. We are currently investigating the distribution and quantity of several oncogene transcripts and gene products in colonic lesions. By using monoclonal antibodies raised against such proteins we can begin to dissect their precise role in the development of human cancer and harness them as tools for diagnosis, prognosis and future therapy.