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Background: PTEN gene at chromosomes 10q23.3 is a tumour suppressor gene that is inactivated in many types of human cancers. The known mechanisms of PTEN inactivation are rendered to mutation, epigenetic silencing by aberrant methylation or gene deletion. Although PTEN role has been documented in many cancers, PTEN alteration in papillary thyroid carcinoma (PTC) has not been fully elucidated. The aim of this study is to comprehensively investigate PTEN alterations in a large cohort of Middle Eastern papillary thyroid cancer by immunohistochemistry and fluorescent in situ hybridisation (FISH)., Methods: PTEN protein expression was analysed by immunohistochemistry in a tissue microarray (TMA) format in a large cohort of more than 1000 patients with papillary thyroid cancer. Copy number changes in PTEN were analysed by FISH and data were correlated with clinicopathological parameters along with survival analysis., Results: PTEN inactivation reflected by complete absence of staining was seen in 24.5% of PTC samples, whereas PTEN deletion was seen only in 4.8% of the tested samples by FISH. No association was seen between PTEN loss of protein expression and PTEN gene deletion. However, interestingly, PTEN loss of expression was significantly associated with the follicular variant subset of papillary thyroid cancer., Conclusion: Our study confirmed that PTEN might have a role in pathogenesis in a subset of PTC. PTEN loss of protein expression is a more common event in follicular variant of papillary thyroid cancer. Lack of association between PTEN loss of protein expression and PTEN gene deletion might indicate that gene deletion may not be the sole cause for PTEN loss of expression and these results might raise the possibility of other mechanism such as promoter methylation-mediated gene silencing to be responsible for PTEN inactivation.

Background: Elevated expression of focal adhesion kinase (FAK) occurs in numerous human cancers including colon-, cervix- and breast cancer. Although several studies have implicated FAK in mammary tumour formation induced by ectopic oncogene expression, evidence supporting a role for FAK in spontaneous mammary tumour development caused by loss of tumour suppressor genes such as p53 is lacking. Alterations in the tumour suppressor gene p53 have been implicated in over 50% of human breast cancers. Given that elevated FAK expression highly correlates with p53 mutation status in human breast cancer, we set out to investigate the importance of FAK in p53-mediated spontaneous mammary tumour development., Methods: To directly assess the role of FAK, we generated mice with conditional inactivation of FAK and p53. We generated female p53(lox/lox)/FAK(+/+)/WapCre, p53(lox/lox)/FAK(flox/+)/WapCre and p53(lox/lox)/FAK(flox/-)/WapCre mice, and mice with WapCre-mediated conditional expression of p53(R270H), the mouse equivalent of human p53(R273H) hot spot mutation, together with conditional deletion of FAK, P53(R270H/+)/FAK(lox/+)/WapCre and p53(R270H/+)/FAK(flox/-)/WapCre mice. All mice were subjected to one pregnancy to induce WapCre-mediated deletion of p53 or expression of p53 R270H, and Fak genes flanked by two loxP sites, and subsequently followed the development of mammary tumours., Results: Using this approach, we show that FAK is important for p53-induced mammary tumour development. In addition, mice with the mammary gland-specific conditional expression of p53 point mutation R270H, the mouse equivalent to human R273H, in combination with conditional deletion of Fak showed reduced incidence of p53(R270H)-induced mammary tumours. In both models these effects of FAK were related to reduced proliferation in preneoplastic lesions in the mammary gland ductal structures., Conclusions: Mammary gland-specific ablation of FAK hampers p53-regulated spontaneous mammary tumour formation. Focal adhesion kinase deletion reduced proliferative capacity of p53 null and p53(R270H) mammary epithelial cells but did not lead to increased apoptosis in vivo. Our data identify FAK as an important regulator in mammary epithelial cell proliferation in p53-mediated and p53(R270H)-induced mammary tumour development.

Oncocytic tumours are characterised by hyperproliferation of mitochondria. We immunohistochemically analysed all enzymes of the oxidative phosphorylation system in 19 oncocytic thyroid tumours. A specific lack of complex I was detected, which was expressed at <5% of the level determined in surrounding non-cancerous tissue.

Manganese superoxide dismutase (SOD2) is an enzyme that catalyses the dismutation of superoxide in the mitochondria, leading to reduced levels of reactive oxygen species. Reduced expression levels of SOD2 have been shown to result in increased DNA damage and sod2 heterozygous mice have increased incidences of cancer. It has also been shown that SOD2 expression is lost in pancreatic cell lines, with reintroduction of SOD2 resulting in decreased rate of proliferation. The mechanism of decreased SOD2 expression in pancreatic carcinoma has not been previously determined. We demonstrate, through sodium bisulphite sequencing, that the sod2 locus is methylated in some pancreatic cell lines leading to a corresponding decrease in SOD2 expression. Methylation can be reversed by treatment with zebularine, a methyltransferase inhibitor, resulting in restored SOD2 expression. Furthermore, we demonstrate that sensitivity of pancreatic carcinoma cell lines to 2-methoxyestradiol correlates with SOD2 expression and SOD2 modulation can alter the sensitivity of these cells. Using both genomics and proteomics, we also identify molecular consequences of SOD2 expression in MIA-PaCa2 cells, including dephosphorylation of VEGFR2 and the identification of both SOD2-regulated genes and transcription factors with altered binding activity in response to SOD2 expression.

Several angiogenic growth factors including fibroblast growth factors 1 and 2 (FGF1 and FGF2) depend on heparan sulphate (HS) for biological activity. We previously showed that all cellular elements in ovarian tumour tissue synthesised HS but biologically active HS (i.e. HS capable of binding FGF2 and its receptor) was confined to ovarian tumour endothelium. In this study, we have sought to explain this observation. Heparan sulphate sulphotransferases 1 and 2 (HS6ST1 and HS6ST2) attach sulphate groups to C-6 of glucosamine residues in HS that are critical for FGF2 activation. These enzymes were strongly expressed by tumour cells, but only HS6ST1 was found in endothelial cells. Immunostaining with the 3G10 antibody of tissue sections pretreated with heparinases indicated that HS proteoglycans were produced by tumour and endothelial cells. These results indicated that, in contrast to the endothelium, HS produced by tumour cells may be modified by cell-surface heparanase (HPA1) or endosulphatase (SULF). Protein and RNA analysis revealed that HPA1 was strongly expressed by ovarian tumour cells in eight of ten specimens examined. HSULF-1, which removes specific 6-O-sulphate groups from HS, was abundant in tumour cells but weakly expressed in the endothelium. If this enzyme was responsible for the lack of biologically active HS on the tumour cell surface, we would expect exogenous FGF2 binding to be preserved; we showed previously that this was indeed the case although FGF2 binding was reduced compared to the endothelium and stroma. Thus, the combined effects of heparanase and HSULF could account for the lack of biologically active HS in tumour cells rather than deficiencies in the biosynthetic enzymes.

Granzyme B (GrB) is the prototypic member of a serine protease family primarily used by cytotoxic lymphocytes to kill target cells. We report here that, by immunohistochemical staining of paraffin-embedded tumour sections, GrB protein was unexpectedly detected in malignant cells of a subset of breast cancers and their adjacent reactive endothelial and mesenchymal cells in which endogenous retinoblastoma protein (pRB) is overexpressed. The identity of the endogenous GrB was further confirmed experimentally in RB-deficient breast carcinoma cell culture upon overexpression of ectopic pRB. Our finding extends the recent paradigm-shifting trend for a more diverse biological role of granzyme B, and might provide a rational basis for exploring its potential prognostic value in a variety of human cancers.

Transformed cells have been documented to be methionine-dependent, suggesting that inhibition of methionine synthesis might be useful for cancer therapy. Methylenetetrahydrofolate reductase synthesises 5-methyltetrahydrofolate, the methyl donor utilised in methionine synthesis from homocysteine by vitamin B(12)-dependent methionine synthase. We hypothesised that methylenetetrahydrofolate reductase inhibition would affect cell viability through decreased methionine synthesis. Using medium lacking methionine, but containing homocysteine and vitamin B(12) (M-H+), we found that nontransformed human fibroblasts could maintain growth. In contrast, four transformed cell lines (one colon carcinoma, two neuroblastoma and one breast carcinoma) increased proliferation only slightly in the M-H+ medium. To downregulate methylenetetrahydrofolate reductase expression, two phosphorothioate antisense oligonucleotides, EX5 and 677T, were used to target methylenetetrahydrofolate reductase in the colon carcinoma line SW620; 400 nM of each antisense oligonucleotide decreased cell survival by approximately 80% (P<0.01) and 70% (P<0.0001), respectively, compared to cell survival after the respective control mismatched oligonucleotide. Western blotting and enzyme assays confirmed that methylenetetrahydrofolate reductase expression was decreased. Two neuroblastoma and two breast carcinoma lines also demonstrated decreased survival following EX5 treatment whereas nontransformed human fibroblasts were not affected. This study suggests that methylenetetrahydrofolate reductase may be required for tumour cell survival and that methylenetetrahydrofolate reductase inhibition should be considered for anti-tumour therapy.

Promoter hypermethylation of CpG islands in tumour suppressor genes can lead to transcriptional inactivation. To investigate the association between methylation and expression at O6-methylguanine-DNA methyltransferase, we performed methylation-specific PCR and immunohistochemistry in 149 gastric carcinomas. Promoter methylation was found in 14.1% of tumours and loss of expression was detected in 11.4% of tumours. To examine correlation between the O6-methylguanine-DNA methyltransferase expression and the clinical data, we investigated O6-methylguanine-DNA methyltransferase expression in 315 consecutive gastric carcinomas. A similar frequency of loss of O6-methylguanine-DNA methyltransferase expression was confirmed in these cases. The loss of O6-methylguanine-DNA methyltransferase expression was significantly associated with pTNM stage (P=0.037), tumour invasion (P=0.02), microsatellite instability (P=0.041) and overall survival (P=0.01). Among 11 gastric cancer cell lines, SNU-620 showed the loss of O6-methylguanine-DNA methyltransferase expression as well as promoter methylation. After treatment with 5-aza-2-deoxycytidine, a demethylating agent, SNU-620 re-expressed O6-methylguanine-DNA methyltransferase mRNA. In summary, we suggest that during gastric carcinogenesis, the loss of O6-methylguanine-DNA methyltransferase expression frequently occurs via the hypermethylation of the CpG islands of the promoter region, and that this is significantly associated with the clinicopathological characteristics., (Copyright 2002 Cancer Research UK)

Apoptosis is of important significance in the pathogenesis of cancer. Many methods are available for the measurement of apoptosis but the 'gold standard' is to identify apoptotic cells by their morphological features using microscopy. Caspase 3 is a cytosolic enzyme that is activated only in cells committed to undergo apoptosis. The activation of caspase 3 precedes the development of the classical morphological features of apoptosis. Using immunohistochemistry with an antibody against the active form of caspase 3, the apoptotic index (AI) was measured in 116 samples of human breast tissue (22 normal/benign and 94 invasive carcinomas). The AI obtained by measuring caspase activation has a strong correlation with the AI derived by morphological assessment (r = 0.736, P < 0.01). The AI is higher in the invasive group than in the benign group (P = 0.008), and in invasive cancer high AI is associated with high tumour grade (P = 0.013), positive node status (P < 0.001) and negative steroid receptor status (P = 0.001 for ER; P = 0.004 for PR). No significant association is observed between AI and tumour size. Measurement of apoptosis by immunohistochemistry using an antibody against the active form of caspase 3 is therefore reliable and correlates strongly with morphological assessment.

We have recently reported a novel function for carbonyl reductase (CR), namely, its ability to modulate the metastatic potential of malignant mouse cells. Because there are currently no data addressing a similar function for CR in human cancers, the aim of this study was to assess a correlation between survival and metastasis, and CR level in epithelial ovarian cancer. Using anti-CR antibody, immunohistochemical staining was performed on 73 epithelial ovarian cancers, 13 borderline malignant tumours, and 25 benign ovarian tumours for a total of 111 specimens. The combined rate for strongly and weakly positive reactions for CR was 32.0% for benign tumours, 38.5% for borderline malignant tumours, and 61.6% for ovarian cancers. The CR-positive rate was 35.7% (weakly positive alone) for ovarian cancers with retroperitoneal lymph node (RLN) metastasis and 67.8% for those without RLN metastasis (P< 0.05). The 5-year survival rate was 62.7% for the patients with CR-negative cancer and 86.1% for those with CR-positive cancer (P< 0.05). The present results indicate that decreased CR expression in epithelial ovarian cancer is associated with RLN metastasis and poor survival., (Copyright 2001 Cancer Research Campaign)

Thymidine phosphorylase (TP) is an essential enzyme for the biochemical activation of 5-fluorouracil (5-FU). Interferon upregulates TP in vivo, although the dose and schedule of interferon for optimal biomodulation of 5-FU is not known. In this study, TP activity was measured in peripheral blood lymphocytes (PBLs) from patients with advanced carcinoma receiving treatment with 5-FU and folinic acid. Cohorts of patients were treated with interferon alpha (IFNalpha), immediately prior to 5-FU/folinic acid, at doses of 3 MIU m(-2), 9 MIU m(-2) and 18 MIUm(-2). IFNalpha was administered on day 0 cycle two, day-1 and day 0 cycle three and day-2, day-1 and day 0 cycle four. A fourth cohort was treated with IFNalpha 9 MIU m(-2) three times per week from cycle 2 onwards. Twenty-one patients were entered into the study with 19 evaluable for response. Six patients (32%) had stable disease and 13 (68%) progressive disease. There were no grade-IV toxicities. TP activity was detected in PBLs from all patients with wide interpatient variability in constitutive TP activity prior to chemotherapy, and in response to IFNalpha. 5-FU/folinic acid alone did not induce TP activity but a single dose of IFNalpha led to upregulation of TP within 2 h of administration with a further increase by 24 h (signed rank test, P = 0.006). TP activity remained elevated for at least 13 days (signed rank test, P= 0.02). There were no significant differences in TP activity between schedules or with additional doses of IFNalpha. A single dose of IFNalpha as low as 3 MIU m(-2) can cause sustained elevation of PBL TP activity in vivo indicating that biochemical markers are important pharmacodynamic endpoints for developing optimal schedules of IFNalpha for biomodulation of 5-FU.

Hyper-activation of mitogen-activated protein kinase (MAPK) has recently been reported in several human cancers and activation of MAPK in those cancers may be associated with carcinogenesis through aberrant cell proliferation. To understand the roles of the MAPK pathway in colorectal tumorigenesis, we examined the status of extracellular signal-regulated protein kinases (ERK1/2) in 21 colorectal tumour specimens and compared it with that of paired normals. The specific MAPK activities were two- to tenfold lower in 71% (15 out of 21 cases) of colorectal tumours compared to those in paired normals. The individual MAPK kinase (MEK) correlated with MAPK activities (P = 0.006). Reduction of the MAPK and MEK activities in colorectal tumours was also observed in adenomas. These results suggested that down-regulation of the MAPK cascade may be caused by early genetic event(s) and that it may be related to the loss of normal growth control. Although MAPK activities were down-regulated both in adenomas and carcinomas, activities of the MAPKs in carcinomas were higher than those of paired adenomas. These results suggested that MAPK activities may be increased in the adenoma-to-carcinoma sequence and that it may play a role in the tumour progression. Observation of the differential regulation of MAPK activities in colorectal tumorigeneis suggested roles for the MAPK pathway in both positive and negative controls of cell growth.

Activation of Met by its ligand HGF has been shown to elicit both mitogenic and motogenic responses in thyrocytes in vitro. In the present study we have investigated the expression of Met in human anaplastic thyroid carcinoma cells in culture. There was a variation in expression level and size of Met in the different cell lines; high Met expression was found in four cell lines, compared to non-neoplastic human thyrocytes. Treatment with glucoproteinase F showed that the size differences observed were due to variances in the degree of glycosylation. Interestingly, in cell lines with high expression of Met, the receptor proteins were found to be constitutively tyrosine phosphorylated. None of these cell lines expressed HGF mRNA, and addition of suramin did not affect the level of tyrosine phosphorylation of Met in unstimulated cells, suggesting the absence of autocrine stimulatory pathways. Furthermore, we did not observe MET gene amplification, activating mutations or phosphatase defects. The tyrosine phosphorylated receptors appeared functionally active since the receptors associated with the adaptor molecule Shc. In summary, we have found ligand-independent constitutively activated Met in four out of six anaplastic thyroid carcinoma cell lines.

Previous studies demonstrated that the immunohistochemical expression of thymidine phosphorylase (dThdPase) was related with distant metastasis and disease progression. In this study we investigated the production of dThdPase/platelet-derived endothelial cell growth factor in gastric cancer quantitatively. In a total of 75 tumour tissues and 60 normal gastric mucosa specimens, dThdPase protein concentrations were determined by ELISA. The amount of dThdPase was significantly higher in the tumour tissue than in the normal tissue. Intratumoural dThdPase concentrations were significantly higher in Borrmann types I and II macroscopically, in poorly differentiated and solid type histologically, in the medullary type of the tumour stroma, and in the tumour-invading serosa. In the medullary type of the amount of tumour stroma, protein levels of dThdPase were positively correlated with the vertical diameter of the tumour (r = 0.580, P = 0.019). By immunohistochemical study, dThdPase expression on tumour cells was observed in all seven specimens with high dThdPase protein levels, but not in all 14 cases with low dThdPase protein levels (P < 0.05). In summary, these data indicated that dThdPase is up-regulated in advanced solid types of gastric cancer, suggesting that dThdPase production in carcinoma cells might be induced by the microenvironment.

Reactive oxygen metabolites are implicated in the initiation and promotion of cancer. In addition, oxidant scavengers, such as manganese--(Mn-SOD) and copper/zinc--superoxide dismutase (Cu/Zn-SOD), are thought to contribute to colorectal cancer treatment response. In the present study, the prognostic significance of the Mn- and Cu/Zn-SOD antigen content of normal mucosa and carcinomas of 163 patients with colorectal cancer was evaluated in comparison with major clinicopathological parameters, with respect to the 5-year overall survival. The Mn-SOD content of carcinomas was found to be significantly higher than that of normal mucosa, whereas there was no difference in the Cu/Zn-SOD content between the normal mucosa and carcinomas. No association was demonstrable between the Mn-SOD and Cu/Zn-SOD content of the tissues and the assessed clinicopathological parameters (gender, age, localization, differentiation grade, diameter and Dukes' stage), with the exception of the Cu/Zn-SOD and the differentiation grade of the carcinomas. Univariate analysis showed that a high Mn-SOD content of carcinomas was associated with a poor 5-year overall survival of the patients with colorectal cancer. Multivariate analysis including all clinicopathological parameters revealed that this Mn-SOD parameter was prognostically independent. The Mn- and Cu/Zn-SOD content of normal mucosa and the Cu/Zn-SOD content of carcinomas were not associated with the overall survival of the patients. In conclusion, this study demonstrates that for patients with colorectal cancer the Mn-SOD content of colorectal carcinomas has a significant prognostic value that is independent from major clinicopathological parameters, including Dukes' stage.

Urokinase plasminogen activator (uPA) is a serine protease involved in cancer invasion and metastasis. uPA acts in vivo by binding to a membrane receptor known as uPAR. In this study, uPA and uPAR levels were semiquantitated by immunocytochemistry in 36 primary breast carcinomas. Using monoclonal antibody HD-UK 1, uPA was detected both in stromal and in malignant cells. However, the predominant location was in the stromal cells. Using double-staining, cells containing uPA were also found to coexpress either cytokeratin (an epithelial cell marker) or more frequently KP1 (a macrophage/monocyte cell marker). With monoclonal antibody HD-uPAR 13.1, uPAR was localized principally to spindle- or macrophage-like stromal cells, especially when these cells surrounded invasive breast cancer. In contrast, uPAR was only rarely detected in cancer cells and was not detected in normal epithelia surrounding tumour or in areas of adenosis. uPA levels in both stromal and epithelial cells were significantly correlated with those for uPAR. We conclude that both uPA and its receptor are mostly present in stromal cells in invasive breast carcinomas. These results suggest that stromal cells collaborate with malignant cells to mediate metastasis.

The high-affinity folate-binding protein (FBP) is primarily involved in the uptake of the 5-methyltetrahydrofolate, and its expression may be physiologically regulated by the intracellular folate content. The overexpression of FBP on the cell surface of ovarian carcinoma cells may be responsible for an increased folate uptake. We tested the hypothesis of the existence of a defect in the 5, 10-methylenetetrahydrofolate reductase (MTHFR) in ovarian tumours that could cause reduced intracellular regeneration of the 5-methyltetrahydrofolate and induce increased FBP expression. No sequence mutations were found in the MTHFR gene, but allelic deletions of this gene were frequently detected in ovarian tumours (59%). Chromosomal losses appeared to be confined to the 1p36.3 region to which the MTHFR gene maps. Although it cannot be stated that MTHFR is the target gene of the chromosomal loss involving the 1p36.3 region, a correlation between loss of heterozygosity at this locus and decrease in MTHFR activity was shown, suggesting a role of these allelic deletions in generating a biochemical defect in folate metabolism. Further studies are needed to assess further the relationship between MTHFR and FBP overexpression, but the demonstration of the alteration of a key metabolic enzyme of the folate cycle in a subset of human ovarian tumours is in accordance with the hypothesis of an altered folate metabolism in these neoplasias and might be exploited for therapeutic purposes.

We have compared the levels of phosphoglycerate mutase, 2,3-bisphosphoglycerate phosphatase and enolase activities and the distribution of their isoenzymes in normal colon, liver and lung tissues, and in colon, liver and lung adenocarcinoma, lung squamous cell carcinoma and lung carcinoid. All tumours presented higher phosphoglycerate mutase and enolase activities and lower 2,3-bisphosphoglycerate phosphatase activity than the normal tissues. No changes were observed in the phosphoglycerate mutase isoenzyme patterns analysed by cellulose acetate electrophoresis. All specimens contained mainly type BB isoenzyme, traces of type MB isoenzyme and no type MM isoenzyme. However, the tumours had decreased levels of 2,3-bisphosphoglycerate mutase and 2,3-bisphosphoglycerate mutase-phosphoglycerate mutase hybrid enzyme. Determined by agarose gel electrophoresis, alpha alpha-enolase was the isoenzyme predominant in normal lung, colon and liver tissue, although alpha gamma- and gamma gamma-enolase were also present in all tissues. In colon, liver and non-endocrine lung tumours, the proportions of alpha gamma- and gamma gamma-enolase decreased. In contrast, in carcinoid tumours of the lung, the proportions of these isoenzymes increased.

We have demonstrated that urinary trypsin inhibitor (UTI) purified from human urine is able to inhibit lung metastasis of mouse Lewis lung carcinoma (3LL) cells in experimental and spontaneous metastasis models. In this study, we have investigated whether UTI in combination with an anti-cancer drug, etoposide, can prevent tumour metastasis and show an enhanced therapeutic effect. Subcutaneous (s.c.) implantation of 3LL cells (1 x 10(6) cells) in the abdominal wall of C57BL/6 female mice resulted in macroscopic lung metastasis within 21 days. Microscopic lung metastasis was established by day 14 after tumour cell inoculation, and surgical treatment alone after this time resulted in no inhibition of lung metastasis. The number of lung tumour colonies in the group of mice which received surgery at day 21 was greater than in mice which had tumours left in situ (P = 0.0017). Surgical treatment on day 7, followed by UTI administration (s.c.) for 7 days, led to a decrease in lung metastasis compared with untreated animals. A significant inhibition of the formation of pulmonary metastasis was obtained with daily s.c. injections of UTI for 7 days immediately after tumour cell inoculation. UTI administration did not affect the primary tumour size at the time of operation. In addition, etoposide treatment alone led to a smaller primary tumours and yielded reduction of the formation of lung metastasis in the group of mice which received surgery at day 14 (P = 0.0026). Even in mice which received surgical treatment on day 14, followed by the combination of UTI (500 micrograms per mouse, days 14, 15, 16, 17, 18, 19 and 20) with etoposide (40 mg kg-1, days 14, 18 and 22), there was significant reduction of the formation of lung metastasis (P = 0.0001). Thus, the combination of an anti-metastatic agent with an anti-cancer drug, etoposide, might provide a therapeutically promising basis for anti-metastatic therapy.

We have evaluated the prognostic value of tumour-associated trypsin inhibitor (TATI) in stage III or IV ovarian cancer. Tumour-associated trypsin inhibitor (TATI) and CA 125 were determined in serum samples from 66 patients taken before primary surgery. TATI was elevated (> 22 micrograms l-1) in 27 patients (41%). These had a 5 year cumulative survival of 8%, whereas survival was 45% in 39 patients with normal preoperative TATI values. By contrast, the preoperative CA 125 level did not predict survival. In multivariate analysis which included age, stage, histological grade and preoperative TATI and CA 125 levels, patients with elevated preoperative TATI levels had a 2.3-fold relative risk of death (95% confidence interval 1.23-4.20; P = 0.002) compared with patients with normal preoperative levels. This result was comparable with the predictive value of primary residual tumour size, since patients with residual tumour larger than 2 cm in diameter had a 5.2-fold relative risk of death (95% confidence interval 2.55-10.68) compared with patients with a smaller or no residual tumour. Thus, preoperative determination of serum TATI may have a place in the pretreatment evaluation of patients with advanced ovarian cancer.

Hormonal regulation of plasminogen activator expression in 7,12-dimethylbenz[a]anthracene (DMBA)--induced rat mammary carcinomas was studied both in vivo and in vitro and was compared to that in DMBA-mammary dysplasia induced in neonatally androgenised rats. The plasminogen activator activity in DMBA-mammary carcinomas, but not in DMBA-mammary dysplasia, was regulated by oestrogen. This suggests that expression of this enzyme is hormonally regulated in carcinoma cells. Furthermore, in two of six DMBA-mammary carcinoma groups classified in terms of hormonal treatment, plasminogen activator activity was not under the control of oestrogen. Thus, the present results suggest that at the time of carcinogenesis, the hormonal milieu determines the hormone sensitivities of the malignant cells.

Using an indirect immunohistochemical technique on paraffin sections, employing a polyclonal antibody to the acidic (placental) form of glutathione-S-transferase (GST), we have evaluated cytoplasmic and nuclear staining in a series of 67 cervical biopsies including normal non neoplastic tissue, immature squamous metaplasia, all grades of cervical intraepithelial neoplasia (CIN) and invasive carcinomas of keratinising and non-keratinising types. No differences in cytoplasmic staining between the varied lesions studied were seen. However, there were marked differences in nuclear staining. While normal non-neoplastic stratified squamous epithelium showed weak staining of the lower one-third of the epithelium only, in immature squamous metaplasia and in all grades of CIN there was intense nuclear staining in all layers of the epithelium. Invasive carcinomas showed generally less intense nuclear staining than CIN lesions. Endocervical cell nuclei also showed intense nuclear staining. These findings indicate that GST is of limited use as a marker of transformation in the human cervix uteri.

Four human head and neck xenograft (HNX) tumour lines grown in nude mice and two murine colon carcinomas (Colon 26 and 38) were tested for their sensitivity to 5-fluorouracil (5-FU) and its prodrug 5'deoxy-5-fluorouridine (Doxifluridine, 5'd-FUR). 5-FU sensitivity at the maximum tolerated dose (MTD) showed the following pattern; HNX-DU less than HNX-KE = HNX-E = HNX-G less than Colon 26 much less than Colon 38. The sensitivity pattern to 5'd-FUR was: HNX-DU less than HNX-G less than HNX-E less than HNX-KE less than Colon 38 less than Colon 26. For HNX-KE, HNX-E and Colon 26 an increase in therapeutic efficacy was observed with 5'd-FUR as compared to 5-FU; Colon 38 was as sensitive to 5'd-FUR as to 5-FU. Plasma pharmacokinetics of 5'd-FUR and 5-FU were comparable in normal and nude mice. Metabolism of 5-FU and 5'd-FUR was studied in the tumours. Conversion of 5'd-FUR to 5-FU was highest in Colon 26 and 15-20 times lower in HNX-DU, HNX-KE and Colon 38. The Km for 5'd-FUR in all tumours was 1-2 mM. Further anabolism of 5-FU to fluorouridine (FUR) was 5-10 times higher than that of 5-FU to FUR in HNX tumours and 3 times in the colon tumours. 5-FU conversion to FUMP via FUR had the following pattern: Colon 26 much greater than HNX-DU greater than HNX-G greater than HNX-E greater than HNX-KE much greater than Colon 38; of 5-FU to FdUMP via FUdR: Colon 26 greater than HNX-DU = HNX-KE greater than HNX-E greater than HNX-G = Colon 38; and that of 5-FU to FUMP catalysed by orotate phosphoribosyl transferase (OPRT); Colon 26 greater than or equal to Colon 38 greater than HNX-KE greater than HNX-E = HNX-DU = HNX-G. Only those tumours with a relatively high activity of OPRT were sensitive to 5'd-FUR. Colon 26, which has a very high rate of pyrimidine nucleoside phosphorylase, showed a relatively high increase in the therapeutic efficacy. It is concluded that a low rate of pyrimidine nucleoside phosphorylase is enough to convert 5'd-FUR to 5-FU; further anabolism of 5-FU catalysed by OPRT may be limiting and explain the differential sensitivity.

The regulation of cholesterol esterification during cell proliferation was studied. The serum free cholesterol, cholesterol esters and lecithin: cholesterol acyltransferase (LCAT) activity of nude mice with and without pancreatic acinar cell tumours and rats with proliferating tissues were determined. In addition, the apparent activity of acyl-CoA: cholesterol acyltransferase (ACAT) in homogenates of nude mouse tumours and proliferating rat tissues were determined and compared with those of normal nude mouse and rat tissues. Serum cholesterol ester levels were significantly lower in host nude mice with tumours and in rats with regenerating liver, and increased significantly in pregnant rats when compared with respective controls. Circulating LCAT activity levels decreased in host nude mice, in pregnant rats, and in rats with regenerating pancreas and regenerating liver. Apparent ACAT activity levels increased significantly in nude mouse tumours and in foetal and postnatal rat pancreata and also in postnatal liver. At the same time, apparent ACAT activity levels decreased in foetal and regenerating rat livers when compared with respective control tissues. These results suggest that serum cholesterol esters, circulating LCAT and cellular ACAT levels are modulated during cell proliferation.