Induction of thymomas by N-methyl-N-nitrosourea in AKR mice: interaction between the chemical carcinogen and endogenous murine leukaemia viruses

AKR mice develop thymomas spontaneously when >6 months oldbut when young AKR mice are treated with N-methyl-N-nitrosourea(MNU) they develop thymomas at 3–6 months of age. In thisstudy the potential role of oncogene activation in the developmentof both the spontaneous and MNU-induced thymomas in AKR micehas been examined by DNA transfection into NTH3T3 mouse fibroblastsand by Southern analysis of tumour DNA. The results show thata high proportion of MNU-induced thymomas contain activatedcellular ras^K while no activated cellular ras genes were detectedin spontaneous thymomas. Southern analysis of tumour DNA revealedthat 2/30 spontaneous tumours and 2/52 MNU-induced tumours containedalterations in the c-myc gene while 5/29 spontaneous tumoursand 6/56 MNU-induced tumours contained alterations in the Pim-1gene. A more detailed analysis of the Pim-1 gene demonstratedthat the alterations observed in most MNU-induced and spontaneoustumours resulted from proviral integration at the 3' end ofthis gene. Our analyses also demonstrated that the majorityof MNU induced tumours, including those containing rearrangementsin the Pim-1 gene, lacked the somatically acquired recombinantMCF proviruses that are present in most spontaneous AKR lymphomas.These results provide evidence that (i) the mechanisms of developmentof MNU-induced and spontaneous tumours in AKR mice are distinctand (ii) the development of thymomas that contain proviral integrationsat the Pim-1 locus in the MNU-treated AKR mice involve cooperationbetween the chemical carcinogen and endogenous murine leukaemiaviruses.     

Expression of carcinoembryonic antigen by adenoma and carcinoma derived epithelial cell lines: possible marker of tumour progression and modulation of expression by sodium butyrate

We have used an indirect immunofluorescence assay to demonstratethe cell membrane expression of carcinoembryonic antigen (CEA)by a pre-malignant colorectal adenoma derived epithelial cellline (PC/AA) and three colorectal carcinoma cell lines (HT29,PC/JW and PC/JW/FI). The results obtained indicated that CEAmay be used as a marker for tumour progression up to the pointof malignant transformation, after which the selection for anaplasticvariants during continuous in vitro culture may result in thesubsequent reduction of cell membrane CEA expression. The percentageof PC/AA cells expressing cell membrane CEA increased from 23.1%of diploid early passage (passage 18) cells to 56.0% of aneuploidlate passage (passage 58) cells. Although non-tumorigenic, theproportion of PC/AA cells expressing cell membrane CEA at latepassage corresponded to that for the PC/JW carcinoma line (56.2%)and is further evidence for the progression of PC/AA in culture.A 3T3 feeder-independent variant of PC/JW (PC/JW/FI) demonstrateda similar percentage of CEA-positive cells as the parental linefor the first 21 passages without feeder support, but by passage27 without 3T3 feeders only 35.3% of cells stained positive.This could be restored to 62.0% by continuous treatment withsodium butyrate (2 mM). A differential growth response to sodiumbutyrate was noted for the pre-malignant adenoma cell line PC/AAand the carcinoma lines HT29 and PC/JW/FI. Concentrations ofsodium butyrate (2 mM) that killed early passage PC/AA cellsallowed the late passage PC/AA cells and the carcinoma linesto proliferate, raising the possibility of sodium butyrate actingas a tumour promotor in the human colorectum.     

Proliferating cell nuclear antigen expression in non-cycling cells may be induced by growth factors in vivo.

The proliferating cell nuclear antigen (PCNA) is required for DNA replication and DNA nucleotide excision repair. Considerable evidence points to PCNA expression being a marker of proliferation in many situations. However, while levels of PCNA are normally very low in non-cycling tissues, high levels of the protein have been observed in the normal tissues surrounding human breast and pancreatic tumours. Using two model systems we have shown that PCNA is induced in non-cycling cells by adjacent transplanted tumour cells and that this phenomenon may be mimicked by the in vivo administration of growth factors (transforming growth factor alpha and epidermal growth factor). These data suggest that tumours may elaborate factors that induce PCNA expression in nearby normal cells. PCNA induction the normal cells surrounding tumours is a direct example of the effect of tumour cells on normal surrounding tissues. This effect may prove to be a useful parameter in the analysis of tumour-host interactions.     

c-Ha-ras not c-Ki-ras activation in three colon tumour cell lines

Using the NIH 3T3 transformation assay system, an activatedc-Ha-ras transforming gene has been identified in three distinctearly passage colon carcinoma cell lines isolated from an invasive,differentiated, adenocarcinoma. The p21 c-Ha-ras gene productfrom these cell lines displayed an altered electrophorecticmobility and a point mutation in the DNA coding sequence leadingto an amino acid substitution at position 12.     

Gene expression in X-irradiated human tumour cell lines expressing cisplatin resistance and altered DNA repair capacity

Previous studies have indicated that excision repair genes,such as ERCC1, or early response genes, such as c-fos, may playa significant role in regulating cellular responses to cisplatin(CDDP) by mediating DNA synthesis and repair pathways. Thispresent study aimed to determine whether altered gene expressionmediated CDDP resistance expressed in two human tumour sublinesfollowing their in vitro exposure to fractionated X-irradiation,not to the drug itself. These sublines, designated SuSa/DXR10and SKOV-3/DXR10, established respectively from a testicularteratoma cell line (SuSa) or an ovarian carcinoma cell line(SKOV-3), expressed stable 3.1- and 2-fold levels of CDDP resistance,as judged by clonogenic assay. Both sublines expressed c-fos,c-myc and thymidylate synthase (TS) RNA constitutively, butat comparable levels to their parental counterparts. Whilstthe ovarian carcinoma cells inherently expressed markedly higherlevels (30- to 50-fold) of the excision repair gene ERCC1 thanthe teratoma cells, only the teratoma DXR10 subline showed anincreased level of expression of ERCC1 mRNA relative to theirparental cells. Expression of the ERCC3/XPB gene encoding arepair helicase, however, was similar in all the lines tested.The results suggest that CDDP resistance may be mediated bydifferent mechanisms in these DXR10 sublines from those previouslyidentified in drug-selected CDDP-resistant human ovarian A2780/DDPcells.     

Cx32 gene mutation in a chemically induced rat liver tumour

Cx32 is a major gap junction protein of the liver and is oftenaberrantly expressed in liver tumours. We have studied mutationof the Cx32 gene during chemically induced hepatocarcinogenesis.DNA from 12 rat liver tumours induced by diethylnitrosamineor N-ethyl-N-hydroxyethylnitrosamine (EHEN) was analysed bythe PCR/SSCP method. One tumour induced by EHEN harboured aGA transition mutation at codon 220, substituting His for Arg.When the mutant DNA was transfected into HeLa cells, which aredeficient in gap junctional intercellular communication (GJIC),GJIC recovered, as in HeLa cells transfected with the wild-typeCx32 gene. Moreover, GJIC was modulated by cAMP, 12-O-tetradec-anoylphorbol-13-acetateand lysophosphatidic acid similarly in mutant and wild-typeCx32 transfectants. These results suggest that Cx32 gene mutationsare rarely involved in rat hepatocarcinogenesis and that themutation found in a tumour may be functionally silent.     

Detection of hypoxic cells in a C3H mouse mammary carcinoma using the comet assay.

The comet assay was used to estimate radiobiological hypoxic fraction across a full range of tumour oxygenations in C3H mammary tumours implanted into the feet of female CDF1 mice. Tumours were either clamped before irradiation or mice were allowed to breath air, 100% oxygen, carbogen or carbon monoxide for 5-35 min before and during exposure to 15 Gy. For the alkaline comet assay, tumours were excised after irradiation and individual tumour cells were analysed for DNA single-strand breaks. Hypoxic cells were defined as those cells with approximately three times fewer single-strand breaks than aerobic cells. Radiobiological hypoxic fraction was calculated by fitting DNA damage histograms to two normal distributions, representing the response of the aerobic and hypoxic populations. The percentage of hypoxic cells estimated using the comet assay was then compared with hypoxic fraction measured using a clamped tumour control assay. Carbogen and oxygen breathing reduced the normal hypoxic fraction from 14% to 2-3% in this tumour, whereas 75-660 p.p.m. carbon monoxide progressively increased the hypoxic fraction from 18% to 82%. The slope of the line comparing the two methods was 1.23 with 95% confidence limits of 1.12-1.33 (r2 = 0.994). In the SCCVII squamous cell carcinoma growing subcutaneously in C3H mice, a similar correlation was observed between hypoxic fraction measured using the comet assay and hypoxic fraction measured in the same tumour cells using the paired survival curve assay (slope = 1.20 with 95% confidence limits of 1.03-1.37). These results confirm the ability of the comet assay to provide an accurate estimate of radiobiological hypoxic fraction over a wide range of tumour oxygenations and between two tumour types.     

Ras mutations in methyiclofenapate-induced B6C3F1 and C57BL/1OJ mouse liver tumours

The majority of genotoxic carcinogen-induced liver tumours ofthe sensitive B6C3F1 mouse contain activated H-ras oncogenes.Such mutations also occur in hepatocarcinogenesis resistantstrains. In order to determine whether this is true of non-genotoxiccarcinogen-induced tumours, liver tumours induced in B6C3F1and C57BL/10J mice by methylclofena pate (MCP) were compared.Polymerase chain reaction (PCR) analysis revealed H-ras codon61 mutations in 11/46 B6C3F1 and 4/31 C57BL/10J liver tumours.The nude mouse tumorigenicity (NMT) assay was used to analysetumours without codon 61 mutations. Of the 12 B6C3F1 liver tumourDNAs subjected to this assay, one contained a H-ras codon 117mutation. Further PCR analysis on frozen tumour samples (46B6C3F1 and 15 C57BL/10J) revealed no codon 12 mutations; oneadditional codon 117 mutation was identified in a B6C3F1 tumour.Overall, then, H-ras codon 61 mutations were detected in MCP-inducedB6C3F1 tumours less frequently than in genotoxin-induced tumours.Two B6C3F1 tumours contained codon 117 mutations similar tothose previously found in tumours induced by ciprofibrate, furanand furfural, and in at least one spontaneous tumour. Ras mutationswere also detected in some C57BL/10J tumours, providing furtherevidence that ras oncogenes can participate in hepatocarcinogenesisin resistant mice.     

Cellular immunity to human urinary bladder carcinoma. I. Correlation to clinical stage and radiotherapy

As tested in vitro in a microcytotoxicity assay, purified peripheral blood lymphocytes from patients with carcinoma of the urinary bladder have a specific destructive effect on primary cultures of autologous and allogeneic tumour target cells, as well as on bladder carcinoma cell lines. No specific effect is observed on cells derived from normal tissues or tumour cells of other histogenic origin. Serial tests made on individual patients have shown that the incidence of lymphocyte cytotoxicity varies with the clinical staging of the disease. While 88% of patients with localized tumours, stage T1-T2, have a response before treatment, the corresponding figure for those with T3-T4 stage tumours is 41%. Local radiotherapy in tumour doses of 3,500–8,400 R either destroyed or diminished existing cytotoxicity. The response remained depressed as long as irradiation was continuous. After therapy all patients with T2 stage tumour redeveloped a response, usually within 7 days. While 80% of T3 patients had cytotoxicity at this time, the incidence in T4 cases was 50%. In some T3 and T4 patients no response redeveloped after radiotherapy or conversely a response was induced by the treatment. Patients lacking cytotoxicity after therapy invariably had distant metastases and/or large residual tumours detected within 3 months. Weak post-therapy cytotoxicity was usually accompanied by development of recurrent tumour or distant metastases during a 9-month observation period; this was not seen in patients whose post-irradiation cytotoxicity was strong during this time. In the absence of viable tumour the post-irradiation response seems to wane with time. Evidence for this comes from patients tested 1-10 years after radiotherapy. Here the presence of cytotoxic lymphocytes was usually associated with viable tumour. The majority of patients clinically tumour free had no response. Maintenance of lymphocyte cytotoxicity, as measured in this system, therefore appears to require the presence of either viable tumour or tumour-derived material. This cytotoxicity is inhibited however by large viable tumours and also metastases.     

Tissue and cell specific methylation, repair and synthesis of DNA in the upper gastrointestinal tract of Wistar rats treated with single doses of N-methyl-N'-nitro-N-nitrosoguanidine

Several potential cancer risk factors have been monitored concurrentlyin the upper gastrointestinal tract of young adult male Wistarrats given single (i.g.) doses of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) which readily induces forestomach tumours under theseconditions. Radioimmunoassay was used to determine the formationof O⁶-methy1-2'-deoxyguanosine (O⁶-MedG) in DNA after MNNG dosesof 1, 5, 25 or 50 mg/kg and was found to be highest in the pylorus,with progressively lower levels in the corpus, forestomach,duodenum, oesophagus and jejunum. Immunohistochemical proceduresshowed that cells with nuclei containing O⁶-MedG were heterogeneouslydistributed in these tissues. O⁶-Alkylguanine-DNA alkyltransferaseactivity in untreated animals was highest in the mucosae ofthe corpus, lower and relatively similar in that of the pylorus,duodenum and jejunum and lowest in the tissues of oesophagusand forestomach. Estimates of DNA synthesis and cell proliferationindicated a 5-fold increase in the DNA labelling index in theforestomach whereas perturbations of DNA synthetic activityin the other tissues of the upper gastrointestinal tract weremuch less marked. As a result of these changes, cells with nucleithat contained O⁶-MedG and were also undergoing DNA synthesis(determined by sequential immunohistochemical analysis and autoradiography)were found most commonly in the forestomach and to a lesserextent in the pylorus. This distribution of replicating damagedcells corresponds with the relative tumour yields in these uppergastrointestinal tract tissues and such cells are the probabletargets in this single dose carcinogenesis regime. Thus, whilstthe highest concentration of O⁶-MedG did not correlate tumourincidence, the overall risk for tumour induction did correlatewith a significant level of DNA damage, a lower capacity forDNA repair and a marked increase in DNA synthesis over the constitutivelevel in the target cells. Carcinogenic risk in this systemis therefore more readily determined by studying several riskfactors simultaneously.     

Cytogenetics of malignant epithelial cells and lymphoblastoid cell lines from nasopharyngeal carcinoma

The malignant epithelial cells of nasopharyngeal carcinoma (NPC) and cells of lines derived form the lymphoid cells which infiltrate this tumour have been investigated cytogenetically. Chromosome spreads of lymphoblastoid cells of lines established from 7 different NPC biopsy specimens were examined after banding staining. Banding was also applied to the epithelial tumour cells of 5 further biopsy specimens freed from non-malignant infiltrating cells by passage through nude mice; epithelial cell spreads were obtained by in vivo splindle arrest. Five of the lymphoblastoid lines were found to be diploid, and 2 tetraploid; the karyotypes were essentially normal. The squamous epithelial nature of the cells in the nude-mouse-grown NPC tumours was established by light and electronmicroscopy, and 3 tumours were found to be near-triploid, and 2 near-diploid. The cells of the near-triploid tumours contained grossly abnormal chromosomes but those of the near-diploid tumours showed only relatively minor changes. Although abnormalities were observed which were specific for cells from each individual tumour, no discernible change was common to cells from all the tumours.     

Cell-cycle distribution of urothelial tumour cells as measured by flow cytometry

The fraction of cells in S + G2 + mitosis from 54 urothelial tumours was calculated by flow cytometry after acridine orange (AO) staining of cells obtained by bladder irrigation or biopsy. Fluorescence signals emitted by the AO-stained DNA and RNA of each cell were separated optically and measured for 5,000 cells per specimen. The patients were classified by the histology of their tumours and clinical data into 5 diagnostic categories: NED (no evidence of disease, but history of bladder tumour), 3; papilloma, 8; non-invasive papillary carcinoma, 8; carcinoma in situ, 17 and invasive carcinoma, 18. The fraction of cells with DNA values in S + G2 + M of the cell cycle varied between 7 and 57% of the total, with a wide range within each diagnostic category, but no statistically significant differences between the groups. The proportion of cells in S + G2 + M from an individual tumour was not correlated with histologic grade or clinical behaviour. The possibility that some tumour cells with DNA values above G1 level are quiescent cells arrested at S or G2 is discussed.     

DNA double-strand break rejoining rates, inherent radiation sensitivity and human tumour response to radiotherapy.

The relationship between DNA double-strand break rejoining rates, inherent radiation sensitivity and tumour response to radiation therapy was determined for a group of 25 squamous cell carcinoma (SCC) and eight sarcoma (SAR) tumours. DNA double-strand break frequencies were measured by neutral filter elution in first passage following explant tumour samples after in vitro exposure to 100 Gy of 60Co gamma-rays. There was no significant difference between SCC and SAR tumour cells in their sensitivity to break induction, but in a 1 h time period SAR tumour cells rejoined significantly fewer breaks than SCC tumour cells, consistent with the greater sensitivity of SAR and suggesting that differences in rates of break rejoining account for the different distributions of radiosensitivities seen when different tumour types are compared. The percentage of breaks rejoined in 1 h in these tumour samples correlated well with D(o) and with the beta component of the survival curve, measured in vitro by clonogenic assay in tumour cell lines established from the tumour samples, but not with SF2 or the alpha component of the survival curve. The rates of DNA double-strand break rejoining therefore appear to influence the exponential portion of survival curves and probably the interactions between breaks. The percentage of breaks rejoined in 1 h was higher in SCC tumours that subsequently failed radiotherapy and, although the differences were not significant, they suggest that rates of break rejoining are an important component of tumour response to radiation therapy.     

Actin polymerisation in Walker 256 carcinoma cells from solid or ascitic tumours

Use was made of the differential DNase I assay to estimate the relative amounts of polymerised and unpolymerised actin in Walker 256 carcinoma cells from solid or ascitic tumours. The concentration of actin per unit DNA and the relative amount of actin present in a polymerised form were both greater in ascitic tumour cells than in cells from solid tumours.     

Rosemary components inhibit benzo[a]pyrene-induced genotoxicity in human bronchial cells

The commonly used spice and flavouring agent, rosemary, derivedfrom the leaves of the plant Rosmarinus officinalis L., displaysantioxidant properties in foods and in biological systems. Moreover,in animal models rosemary components were found to inhibit theinitiation and tumour promotion phases of carcinogenesis. Inthis work, we studied the mechanisms by which rosemary componentsblock initiation of carcinogenesis by the procarcinogen benzo[a]pyrene(B[a]P) in human bronchial epithelial cells (BEAS-2B). Wholerosemary extract (6 µg/ml) or an equivalent concentrationof its most potent antioxidant constituents, carnosol or carnosicacid, inhibited DNA adduct formation by 80% after 6 h co-incubationwith 1.5 µM B[a]P. Under similar conditions, cytochromeP450 (CYP) 1A1 mRNA expression was 50% lower in the presenceof rosemary components, and CYP1A1 activity was inhibited 70–90%.The observed reduction of DNA adduct formation by rosemary componentsmay mostly result from the inhibition of the activation of benzo[a]pyreneto its ultimate metabolites. Carnosol also affected expressionof the phase II enzyme glutathione-S-transferase which is knownto detoxify the proximate carcinogenic metabolite of B[a]P.Treatment of BEAS-2B cells with carnosol (1 µg/ml) for24 h resulted in a 3- to 4-fold induction of GST     

Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86

Background:

Radiotherapy is central in the treatment of cervical cancer. The formation of DNA double-strand breaks is considered to be critical for the radiotherapeutic effect. The non-homologous end joining (NHEJ) proteins DNA–PKcs, Ku70 and Ku86 have a major role in repairing DNA lesions. The objective of this study was to analyse if the expression of DNA–PKcs, Ku70 and Ku86 and their downstream signalling molecules p53, p21 and Mdm-2 are altered in residual cervical tumours after radiotherapy.

Methods:

Retrospective analysis of 127 patients with cervical cancer stage IB-IIA treated with preoperative radiotherapy and radical surgery, revealed residual tumour in the cervical specimen in 30 patients. In 22 cases tumour material from residual and corresponding primary tumour were retrieved and the expression of DNA–PKcs, Ku86, Ku70, p53, p21 and Mdm-2 were assessed by immunohistochemistry.

Results:

Residual tumours showed increased frequency of DNA–PKcs (P=0.037), Ku70 (P=0.018), Ku86 (P=0.008) positive cells. A correlation in DNA–PKcs expression between primary and residual tumours was found. The frequency of p21-positive cells was decreased (P=0.007) in residual tumours whereas no change in p53 or Mdm-2-positive cells were observed.

Conclusion:

Our results show that cervical carcinoma surviving radiotherapy have an increased DNA–PK expression. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of DNA–PK function may be part of a radioresistance mechanism within this tumour type.     

The behaviour of carcinoma of the large bowel in man following transplantation into immune deprived mice

The growth of carcinoma of the human large bowel was studied in the first 2 passages in immune deprived mice. The tumours were obtained from large bowel resections on 3 people. There was a strong histological similarity between the patient''s tumour and the tumour that grew subcutaneously in the mice 2-8 months after implantation. One dissimilarity observed was a higher mitotic index in some of the tumours growing in the immune deprived animals. In the second passage of the bowel tumours, cells were implanted into groups of 8-10 animals in the following sites: subcutaneous, intramuscular, intravenous, intrahepatic, intraperitoneal and intrathoracic. Growth of tumour was observed from all 3 tumours when they were implanted subcutaneously, intramuscularly, intraperitoneally and intrathoracically. Infiltration of muscle by tumour was a frequent finding. Lung metastases developed after intravenous injection of cells in 1 of the 3 tumours. In none of the 3 tumours did growth follow injection of cells directly into the substance of the liver. On no occasions were spontaneous metastases observed.     

Ha-rasVa112 but not p53Ser247 leads to a significant neoplastic transformation rate of the putative rat liver stem cells (oval cell)

In order to test the controversially discussed hypothesis thatoval cells are part of a liver stem cell compartment and cangive rise to cholangiocellular as well as hepatocellular carcinomasin the course of liver carcinogenesis, we trans-fected an ovalcell line established in our laboratory with an oncogenicallyactivated genomic Ha-ras clone (pUC EJ 6.6), carrying a valineat position 12 instead of the wild-type glycine, or a rat p53cDNA mutated by site-directed mutagenes is at codon 247, whichcorresponds to codon 249 in the human p53. This codon is ofparticular interest since it represents a mutation hotspot observedin hepatocellular carcinoma especially in regions with highaflatoxin B₁ exposure. Independent Ha-ras^Val112Wain and p53Ser247recombinant clones were subcutaneously injected into syngeneicnewborn rats and the resulting tumours were analysed histopathologically.Each of two p53^Ser247 clones gave negligible tumour yields (onetumour out of 13 injected animals), whereas each of two Ha-ras^Val12clones gave marked tumour yields (four tumours out of 13 andseven out of 12 treated animals, respectively). In addition,the p53^Ser247-induced tumours appeared only after 11 monthsand were small, whereas the Ha-ras-induced tumours appearedalready after 6–8 weeks and grew rapidly. Histopathologicalanalysis of the tumours revealed only undifferentiated carcinomas.Interestingly, one tumour that arose upon injection of Ha-ras^Val12-transfectedcells stained positive for albumin, showing at least a partialhepatocytic differentiation.