Maintenance of active chromatin states by HMGN2 is required for stem cell identity in a pluripotent stem cell model.

BACKGROUND: Members of the HMGN protein family modulate chromatin structure and influence epigenetic modifications. HMGN1 and HMGN2 are highly expressed during early development and in the neural stem/progenitor cells of the developing and adult brain. Here, we investigate whether HMGN proteins contribute to the chromatin plasticity and epigenetic regulation that is essential for maintaining pluripotency in stem cells. RESULTS: We show that loss of Hmgn1 or Hmgn2 in pluripotent embryonal carcinoma cells leads to increased levels of spontaneous neuronal differentiation. This is accompanied by the loss of pluripotency markers Nanog and Ssea1, and increased expression of the pro-neural transcription factors Neurog1 and Ascl1. Neural stem cells derived from these Hmgn-knockout lines also show increased spontaneous neuronal differentiation and Neurog1 expression. The loss of HMGN2 leads to a global reduction in H3K9 acetylation, and disrupts the profile of H3K4me3, H3K9ac, H3K27ac and H3K122ac at the Nanog and Oct4 loci. At endodermal/mesodermal genes, Hmgn2-knockout cells show a switch from a bivalent to a repressive chromatin configuration. However, at neuronal lineage genes whose expression is increased, no epigenetic changes are observed and their bivalent states are retained following the loss of HMGN2. CONCLUSIONS: We conclude that HMGN1 and HMGN2 maintain the identity of pluripotent embryonal carcinoma cells by optimising the pluripotency transcription factor network and protecting the cells from precocious differentiation. Our evidence suggests that HMGN2 regulates active and bivalent genes by promoting an epigenetic landscape of active histone modifications at promoters and enhancers.

FOXM1 and polo-like kinase 1 are co-ordinately overexpressed in patients with gastric adenocarcinomas.

BACKGROUND: Gastric cancers present late in life with advanced disease and carry a poor prognosis. Polo-like Kinase 1 (PLK1) is a mitotic kinase with regulatory functions during G2/M and mitosis in the cell cycle. In mammalian cells, there is an intricate co-regulatory relationship between PLK1 and the forkhead transcription factor FOXM1. It has been demonstrated that individually either PLK1 or FOXM1 expression predicts poorer survival. However, the co-expression of both of these markers in gastric adenocarcinomas has not been reported previously. METHODS: We aimed to assess the expression of PLK1 and FOXM1 in Gastric adenocarcinomas in a Western Population, to examine whether there is a relationship of PLK1 to FOXM1 in cancer samples. We assess both the protein and mRNA expression in this patient population by Tissue Microarray immunohistochemistry and RT-PCR. RESULTS: Immunohistochemistry was performed on biopsy samples from 79 patients with gastric cancer. Paired normal controls were available in 47 patients. FOXM1 expression was significantly associated with gastric adenocarcinoma (p = 0.001). PLK1 and FOXM1 co-expression was demonstrated in 6/8 (75 %) tumours when analysed by RT-PCR. FOXM1 is overexpressed in a large proportion of gastric carcinomas at the protein level and FOXM1 and PLK1 are concomitantly overexpressed at the mRNA level in this cancer type. CONCLUSIONS: This study has demonstrated that FOXM1 and its target gene PLK1 are coordinately overexpressed in a proportion of gastric adenocarcinomas. This suggests that chemotherapeutic treatments that target this pathway may be of clinical utility.

Expression of hypoxia-inducible factor-1α predicts benefit from hypoxia modification in invasive bladder cancer.

BACKGROUND: The addition of carbogen and nicotinamide (CON) to radiotherapy (RT) improves overall survival in invasive bladder cancer. We explored whether expression of the hypoxia marker hypoxia-inducible factor-1α (HIF-1α) alone or in combination with other markers predicted benefit from CON. METHODS: A retrospective study was carried out using material from patients with high-grade invasive bladder carcinoma enrolled in the BCON phase III trial of RT alone or with CON (RT+CON). HIF-1α expression was studied in 137 tumours using tissue microarrays and immunohistochemistry. Data were available from other studies for carbonic anhydrase IX and glucose transporter 1 protein and gene expression and tumour necrosis. RESULTS: Patients with high HIF-1α expression had improved 5-year local relapse-free survival with RT+CON (47%) compared with RT alone (21%; hazard ratio (HR) 0.48, 95% CI 0.26-0.8, P=0.02), no benefit was seen with low HIF-1α expression (HR 0.81, 95% CI 0.43-1.50, P=0.5). Combinations of markers including necrosis also predicted benefit but did not improve on prediction using necrosis alone. CONCLUSIONS: HIF-1α may be used to predict benefit from CON in patients with bladder cancer but does not improve on use of necrosis.

Investigation of the epithelial to mesenchymal transition markers S100A4, vimentin and Snail1 in gastroesophageal junction tumors.

Epithelial to mesenchymal transition (EMT) promotes tumor progression and invasion. As no study has focused on gastroesophageal junction (GEJ) tumors, the expression of three EMT-related proteins (S100A4, vimentin, and Snail1) was investigated with the aim of assessing their pathologic and prognostic significance. Resection specimens were obtained from 104 patients who underwent surgery for GEJ adenocarcinoma, without preoperative chemotherapy. Three tissue cores were obtained from each of the tumor body (TB), luminal surface (LS), and invasive edge (IE) to produce tissue microarrays, and immunohistochemical staining was performed. The microarrays were scored independently by two observers. The demographic and histopathologic details of the patients were collected. Overall positive expression was observed in 88 (S100A4, 85%), 16 (vimentin, 14%), and 92 (Snail1, 89%) tumors. Staining for S100 A4 was positive in 79 (76%) of TB, 69 (66%) of IE, and 69 (66%) of LS specimens. Staining for vimentin was positive in 7 (6%) of TB, 11 (11%) of IE, and 5 (5%) of LS specimens. Staining for Snail1 was positive in 83 (80%) of TB, 51 (49%) of IE, and 78 (75%) of LS specimens. Positive staining of TB for S100A4 (P = 0.04) and Snail1 at IE (P = 0.01) was associated with involvement of circumferential resection margins. Positive staining for S100A4 in the TB (P = 0.02) and LS (P = 0.01) was associated with poor 5-year overall survival. Vimentin had no statistically significant relationships with pathologic factors or outcome. The acquisition of mesenchymal protein S100A4 is associated with a poor prognosis in patients with GEJ tumors who undergo potentially curative surgery, and LS samples can be used to obtain prognostic information. Increased EMT-related protein expression (S100A4, Snail1) is associated with the involvement of circumferential resection margin.

Lack of prognostic effect of carbonic anhydrase-9, hypoxia inducible factor-1α and bcl-2 in 286 patients with early squamous cell carcinoma of the glottic larynx treated with radiotherapy.

AIMS: To evaluate the prognostic significance of potential tumour markers of hypoxia and apoptosis in early squamous cell carcinoma of the glottic larynx managed with radiotherapy. MATERIALS AND METHODS: In total, 382 patients with T1 and T2 squamous cell carcinoma of the glottic larynx (vocal cords) received radical radiotherapy (50-55 Gy, in 16 fractions in 98% of cases). Pre-treatment haemoglobin was available for 328 patients; biopsy samples were available for 286. Immunohistochemistry was carried out for carbonic anhydrase-9 (CA-9), hypoxia inducible factor-1α (HIF-1α) and Bcl-2. RESULTS: At 5 years, locoregional control was achieved in 88.2%, cancer-specific survival in 95.0% and overall survival in 78.7%. Adverse prognostic factors for locoregional tumour recurrence were pre-treatment haemoglobin <13.0 g/dl (P = 0.035, Log rank test; sensitivity 0.28, specificity 0.84) and stage T2 rather than T1 (P = 0.002). The effect of haemoglobin level on locoregional control was not significant when stratified by the median of 14.2 g/dl (P = 0.43) or as a continuous variable (P = 0.59). High CA-9 (P = 0.11), HIF-1α (P = 0.67) and Bcl-2 (P = 0.77) expression had no prognostic significance. CONCLUSIONS: High CA-9, HIF-1α and Bcl-2 do not add to the prognostic significance of tumour stage and lower haemoglobin in predicting failure of local control in early glottic larynx squamous cell carcinoma managed with radiotherapy. The effect of haemoglobin was not strong enough to be useful as a prognostic biomarker.

The FOXM1-PLK1 axis is commonly upregulated in oesophageal adenocarcinoma.

BACKGROUND: The transcription factor FOXM1 is an important regulator of the cell cycle through controlling periodic gene expression during the G2 and M phases. One key target for FOXM1 is the gene encoding the protein kinase PLK1 and PLK1 itself acts in a positive feedback loop to phosphorylate and activate FOXM1. Both FOXM1 and PLK1 have been shown to be overexpressed in a variety of different tumour types. METHODS: We have used a combination of RT-PCR, western blotting, tissue microarrays and metadata analysis of microarray data to study whether the FOXM1-PLK1 regulatory axis is upregulated and operational in oesophageal adenocarcinoma. RESULTS: FOXM1 and PLK1 are expressed in oesophageal adenocarcinoma-derived cell lines and demonstrate cross-regulatory interactions. Importantly, we also demonstrate the concomitant overexpression of FOXM1 and PLK1 in a large proportion of oesophageal adenocarcinoma samples. This co-association was extended to the additional FOXM1 target genes CCNB1, AURKB and CKS1. In a cohort of patients who subsequently underwent surgery, the expression of several FOXM1 target genes was prognostic for overall survival. CONCLUSIONS: FOXM1 and its target gene PLK1 are commonly overexpressed in oesophageal adenocarcinomas and this association can be extended to other FOXM1 target genes, providing potentially important biomarkers for predicting post-surgery disease survival.

Tissue factor expression in the metaplasia-adenoma-carcinoma sequence of gastric cancer in a European population.

BACKGROUND: Tissue factor (TF), which has a role in normal tissue haemostasis, was reported to be aberrantly expressed, associated with higher microvascular density and a poor prognosis in intestinal-type gastric adenocarcinoma in the Japanese population. This is the first study to look at the relationship of TF and the metaplasia-adenoma-carcinoma sequence (MACS) of gastric cancer in a European population. METHODS: The expression of TF was examined immunohistochemically in 191 gastric tissue samples: (13: normal; 18: intestinal metaplasia; 160: gastric adenocarcinoma) from the European population. RESULTS: TF was not expressed in normal gastric mucosal cells. A strong intensity of staining was found in intestinal metaplasia cells but in 2 of 18 samples. TF expression increased with advancing stage of gastric cancer (P<0.0001, Jonckheere's test for ordered medians). Stage 3-4 gastric cancers preferentially expressed TF (34%, P=0.04). In comparison with the Japanese study, TF was not expressed at a higher level in intestinal vs diffuse-type gastric cancers and expression had 'no prognostic' significance. CONCLUSION: TF may be involved in tumour progression along the MACS of gastric cancer in the European population and is shown to start in precancerous lesions. However, clinical features may differ due to differences in biological features in the two populations, as reflected by differences in TF expression profile.

Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation.

BACKGROUND: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. METHODS: Bladder (n=140, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. RESULTS: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=-0.30, P<0.01) and cervix (R=-0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10(-5)). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). CONCLUSION: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Use of multiple biological markers in radiotherapy-treated head and neck cancer.

OBJECTIVE: Management of patients with head and neck squamous cell carcinoma is often based on clinical parameters, with little appreciation of the underlying tumour biology. Single biological marker studies fail to acknowledge the complexity of these tumours. Our aim was to define a profile of biological markers associated with outcome. DESIGN: This retrospective study involved consecutive patients with oropharyngeal squamous cell carcinoma treated with primary radiotherapy between 1996 and 2001. Pre-treatment biopsies were used to study the immunohistochemical expression of nine biological markers. Markers were chosen to reflect biologically relevant pathways. RESULTS: Following analysis of nine markers, a profile of two markers was derived (carbonic anhydrase 9 and major vault protein), the co-expression of which conferred a significantly poor probability of locoregional control. The prognostic effect of these biomarkers in combination was greater than their effect individually. CONCLUSION: Biomarker profiles can be established which highlight large differences in locoregional control. Identifying tumours that express both carbonic anhydrase 9 and major vault protein may facilitate patient selection for more aggressive treatment.

Hypoxia-associated markers in gastric carcinogenesis and HIF-2alpha in gastric and gastro-oesophageal cancer prognosis.

The study investigated hypoxia-associated markers (HIF-2alpha, Epo, Epo-R, Glut-1 and VEGF) along with Ki-67 in a gastric carcinogenesis model, and the prognostic significance of hypoxia-inducible factor (HIF)-2alpha in surgically treated gastro-oesophageal cancer. Protein expression was examined using immunohistochemistry on formalin-fixed, paraffin-embedded biopsies of normal mucosa (n=20), Helicobacter pylori-associated gastritis (n=24), intestinal metaplasia (n=24), dysplasia (n=12) and intestinal (n=19) and diffuse (n=21) adenocarcinoma. Relationships between HIF-2alpha expression and prognosis were assessed in resection specimens from 177 patients with gastric and gastro-oesophageal junction adenocarcinoma. Expression of all markers increased with progression along the gastric carcinogenesis sequence (P=0.0001). Hypoxia-inducible factor-2alpha was expressed in 63% of 177 resection specimens and at a high level in 44%. The median overall survival in patients with HIF-2alpha-expressing tumours was 22 (95% CI 18-26) months, whereas those with HIF-2alpha-negative tumours had a median survival of 37 (95% CI 29-44) months (P=0.015). Hypoxia-inducible factor-2alpha had no independent prognostic significance in multivariate analysis. In view of the lack of independent prognostic significance, HIF-2alpha has no role as a routine prognostic indicator. However, the high expression of HIF-2alpha suggests that it may be of value as a potential therapeutic target.

Increasing expression of hypoxia-inducible proteins in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence.

Hypoxia-associated markers are involved in the progression of several malignancies, but are relatively unstudied in Barrett's carcinogenesis. Our aim was to assess the immunohistochemical expression of hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, erythropoietin (Epo), Epo receptor (Epo-R), Glut-1 and vascular endothelial growth factor (VEGF) along with Ki67/MIB-1 in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence. Endoscopic biopsies of normal squamous epithelium (NSE) (n=20), columnar-lined oesophagus (CLO) (n=15), CLO with intestinal metaplasia (n=20), dysplasia (n=17) and Barrett's type adenocarcinoma (n=20) were obtained. Immunohistochemistry was performed on the paraffin-embedded tissue. A score was calculated for each marker (range 0-300) by multiplying intensity (none 0, weak 1, moderate 2, strong 3) by percentage of expression (range 0-100). Significant increases in the expression of HIF-2alpha (P=0.014), VEGF (P<0.0001), Epo-R (P<0.0001) and Ki67 (P<0.0001) were found as tissue progressed from NSE to adenocarcinoma. HIF-2alpha was expressed late in the sequence and was only seen in dysplasia and adenocarcinoma. High HIF-2alpha expression was seen in 12 out of 20 Barrett's type adenocarcinoma. The late expression of HIF-2alpha in the Barrett's carcinogenesis sequence and its high expression in adenocarcinoma suggest that it is worth further investigation as a marker of disease progression and therapeutic target.

Hypoxia-inducible factor-1alpha expression in the gastric carcinogenesis sequence and its prognostic role in gastric and gastro-oesophageal adenocarcinomas.

Hypoxia-inducible factor-1 (HIF-1)alpha expression was studied in the gastric carcinogenesis sequence and as a prognostic factor in surgically resected gastric and gastro-oesophageal junction tumours. Protein expression was examined using immunohistochemistry on formalin-fixed biopsies of normal mucosa (n=20), Helicobacter pylori associated gastritis (n=24), intestinal metaplasia (n=24), dysplasia (n=12) and intestinal (n=19) and diffuse (n=21) adenocarcinoma. The relationship between HIF-1alpha expression and prognosis was assessed in resection specimens from 177 patients with gastric and gastro-oesophageal junction adenocarcinoma. Hypoxia-inducible factor-1alpha expression was not observed in normal gastric mucosa but increased in density (P<0.01) and intensity (P<0.01) with progression from H. pylori-associated gastritis, intestinal metaplasia, dysplasia to adenocarcinoma. The pattern of staining in the resection specimens was focally positive in 49 (28%) and at the invasive tumour edge in 41 (23%). Invasive edge expression was associated with lymph node metastases (P=0.034), advanced TNM stage (P=0.001) and was an adverse prognostic factor for cancer-specific survival (P=0.019). In univariate analysis and in comparison with tumours not expressing HIF-1alpha, invasive edge staining was associated with a hazard ratio of 1.6 (95% CI 1.0-2.5) and focally positive staining a hazard ratio of 0.7 (95% CI 0.5-1.2). Hypoxia-inducible factor-1alpha lost prognostic significance in multivariate analysis. The results suggest HIF-1alpha is involved in gastric carcinogenesis and disease progression, but is only a weak prognostic factor for survival.

Expression of the proapoptotic protein Bid is an adverse prognostic factor for radiotherapy outcome in carcinoma of the cervix.

The Bcl-2 family of apoptotic regulators is thought to play an essential role in cancer development and influence the sensitivity of tumour cells to radiotherapy. Bid is an abundantly expressed Bcl-2 family protein playing a central role in various pathways of apoptosis by integrating and converging signals at the mitochondria. The relevance of apoptotic modulation by Bcl-2 and related proteins in tumour development and radiation response for human tumours remains undefined. Therefore, a study was made regarding the expression of Bid in patients with locally advanced cervix carcinoma who received radiotherapy. Bid expression was assessed using immunohistochemistry in pretreatment archival biopsies from 98 patients. The data were correlated with clinicopathologic characteristics and treatment outcome. Pretreatment tumour radiosensitivity data were available for 60 patients. Strong Bid expression was associated with a patient age less than the median of 52 years (P=0.034) and poor metastasis-free survival. In multivariate analysis, after allowing for stage, Bid expression was a significant prognostic factor for both disease-specific and metastasis-free survival (P=0.026). It is concluded that strong tumour Bid expression is associated with poor outcome following radiotherapy regardless of intrinsic tumour cell radiosensitivity, and is adverse prognostic for disease-specific and metastasis-free survival in younger patients.

Cytotoxicity of the bioreductive agent RH1 and its lack of interaction with radiation.

BACKGROUND AND PURPOSE: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions. PATIENTS AND METHODS: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h. RESULTS: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions. CONCLUSIONS: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.

Characterizing the influence of structure and route of exposure on the disposition of dithiocarbamates using toluene-3,4-dithiol analysis of blood and urinary carbon disulfide metabolites.

Differences in the toxicities observed for dithiocarbamates have been proposed to result from the influence of nitrogen substitution, oxidation state, and route of exposure. To better characterize the fate of dithiocarbmates in vivoas a function of structure and route of exposure, rats were administered equimolar doses of carbon disulfide (CS2), N-methyldithiocarbamate, pyrrolidine dithiocarbamate, N,N-diethyldithiocarbamate, or disulfiram daily for five days, either po or ip, and sequential blood samples obtained. Protein dithiocarbamates formed by the in vivo release of CS2, parent dithiocarbamate, and protein-bound mixed disulfides were assessed in plasma and hemolysate by measuring toluene trithiocarbonate generated upon treatment with toluene-3, 4-dithiol (TdT). To aid in determining the bioavailability of CS2 from the administered dithiocarbamates, the urinary CS2 metabolites, 2-thiothiazolidine-4-carboxylic acid (TTCA) and 2-thiothiazolidin-4-ylcarbonylglycine (TTCG), were also determined. The levels of TdT-reactive moieties detected depended upon both the compound administered and the route of exposure. Parent dithiocarbamates, with the exception of disulfiram, were eliminated from blood within 24 h; but protein associated TdT-reactive moieties persisted and accumulated with repeated exposure, regardless of the route of exposure. N-Methyldithiocarbamate demonstrated the greatest potential to produce intracellular globin modifications, presumably through its unique ability to generate a methylisothiocyanate metabolite. Urinary excretion of TTCA and TTCG was more sensitive than TdT analysis for detecting dithiocarbamate exposure, but TdT analysis appeared to be a better indicator of in vivo release of CS2 by dithiocarbamates than were urinary CS2 metabolites. These data suggest that CS2 is a more important metabolite, following oral exposure, than are other routes of exposure, e.g., inhalation or dermal. In addition, data also suggest that acid stability, nitrogen substitution, and route of exposure are important factors governing the toxicity observed for a particular dithiocarbamate.

Glucose transporter-1 (GLUT-1): a potential marker of prognosis in rectal carcinoma?

The aim of the study is to evaluate the pattern and level of expression of glucose transporter-1 (GLUT-1) in rectal carcinoma in relation to outcome as a potential surrogate marker of tumour hypoxia. Formalin-fixed tumour sections from 43 patients with rectal carcinoma, who had undergone radical resection with curative intent, were immunohistochemically stained for GLUT-1. A mean of three sections per tumour (range 1-12) were examined. Each section was semiquantitatively scored; 0, no staining; 1, <10%; 2, 10-50%; 3, >50% and a score given for the whole section, the superficial (luminal) and deep (mural) part of the tumour. Staining was seen in 70% of tumours. Increased staining was noted adjacent to necrosis and ulceration. A diffuse and patchy pattern of staining, with and without colocalisation to necrosis was seen. Patients with high GLUT-1-expressing tumours (score 3 vs 0-2) had a significantly poorer overall survival (P=0.041), which was associated with poorer metastasis-free survival with no difference in local control. No significant correlation was seen with other prognostic factors. There was a strong correlation between the score for the superficial and deep parts of the tumour (r=0.81), but a significant relationship with outcome was only found in the deep part (P=0.003 vs P=0.46). In conclusion, increased GLUT-1 expression in rectal tumours was an adverse prognostic factor and is worth further evaluation as a predictive marker of response to therapy.

The ratio of NPP to GPP: evidence of change over the course of stand development.

Using Scots pine (Pinus sylvestris L.) in Fenno-Scandia as a case study, we investigate whether net primary production (NPP) and maintenance respiration are constant fractions of gross primary production (GPP) as even-aged mono-specific stands progress from initiation to old age. A model of the ratio of NPP to GPP is developed based on (1) the classical model of respiration, which divides total respiration into construction and maintenance components, and (2) a process-based model, which derives respiration from processes including construction, nitrate uptake and reduction, ion uptake, phloem loading and maintenance. Published estimates of specific respiration and production rates, and some recent measurements of components of dry matter in stands of different ages, are used to quantify the two approaches over the course of stand development in an average environment. Both approaches give similar results, showing a decrease in the NPP/GPP ratio with increasing tree height. In addition, we show that stand-growth models fitted under three different sets of assumptions-(i) annual specific rates of maintenance respiration of sapwood (mW) and photosynthesis (sC) are constant; (ii) m(W) is constant, but sC decreases with increasing tree height; and (iii) total maintenance respiration is a constant fraction of GPP and s(C) decreases with increasing tree height-can lead to nearly identical model projections that agree with empirical observations of NPP and stand-growth variables. Remeasurements of GPP and respiration over time in chronosequences of stands may be needed to discern which set of assumptions is correct. Total (construction + maintenance) sapwood respiration per unit mass of sapwood (kg C (kg C year)-1) decreased with increasing stand age, sapwood stock, and average tree height under all three assumptions. However, total sapwood respiration (kg C (ha year)-1) increased over the course of stand development under (i) and (ii), contributing to a downward trend in the time course of the NPP/GPP ratio after closure. A moderate decrease in mW with increasing tree height or sapwood cross-sectional area had little effect on the downward trend. On the basis of this evidence, we argue that a significant decline in the NPP/GPP ratio with tree size or age seems highly probable, although the decline may appear insignificant over some segments of stand development. We also argue that, because stand-growth models can give correct answers for the wrong reasons, statistical calibration of such models should be avoided whenever possible; instead, values of physiological parameters should come from measurements of the physiological processes themselves.

Identification of a new urinary metabolite of carbon disulfide using an improved method for the determination of 2-thioxothiazolidine-4-carboxylic acid.

A new method is reported for the analysis of 2-thioxothiazolidine-4-carboxylic acid (TTCA) in urine that is amenable to automation and provides greatly simplified chromatograms. The method comprises the addition of tetrahydro-2-thioxo-2H-1,3-thiazine-4-carboxylic acid, which is chemically similar to TTCA, as internal standard, purification on an Oasis HLB solid-phase extraction column, and analysis by HPLC with UV detection. The limit of detection for TTCA was 40 pmol/mL of urine, recovery was 79.3 +/- 1.0%, and detection was linear over at least 3 orders of magnitude. In addition, during the analysis of urine samples from workers exposed to CS(2), a novel urinary metabolite of CS(2) was recognized. The new metabolite demonstrated a dose response, was present at approximately 30% the level of TTCA, and was charaterized to be 2-thioxothiazolidin-4-ylcarbonylglycine (TTCG). Administration of TTCG to rats resulted in excretion of TTCA suggesting that TTCG is a likely precursor of TTCA. Although urinary excretion of both TTCA and TTCG resulted from administration of captan, only TTCA was detected following administration of methyl isothiocyanate. The greater selectivity of TTCG suggests that co-analysis of TTCA and TTCG in urine may aid in differentiating exposures to CS(2), captan and isothiocyanates.

Lymphocyte radiosensitivity is a significant prognostic factor for morbidity in carcinoma of the cervix.

PURPOSE: To study the relationship between pretreatment peripheral blood lymphocyte radiosensitivity and morbidity following radiation therapy. METHODS AND MATERIALS: A prospective study was carried out in which patients with carcinoma of the cervix underwent radiation therapy. Intrinsic radiosensitivity was measured on pretreatment peripheral blood lymphocytes, using a limiting dilution clonogenic assay. Late morbidity was assessed using the Franco-Italian glossary. Results were correlated in an actuarial analysis. RESULTS: There were no correlations between the measured lymphocyte radiosensitivity (SF2) and colony-forming efficiency, patient age, tumor grade, or disease stage. For 83 patients, lymphocyte SF2 was a significant prognostic factor for the probability of developing both any (p = 0.002) and Grade 3 (p = 0.026) morbidity. In 174 patients, stage showed borderline significance as a prognostic factor for morbidity (p = 0.056). However, the type of treatment (intracavitary alone, intracavitary plus parametrial irradiation, single insertion plus whole-pelvis irradiation) was significantly associated with the probability of developing late complications (p = 0.013). There was a weak significant inverse correlation between lymphocyte SF2 and grade of morbidity (r = -0.34, p = 0.002). CONCLUSION: These data highlight the importance of normal cell radiosensitivity as a factor determining radiation therapy response. They also show that peripheral blood lymphocyte SF2 is a highly significant prognostic factor for the probability of developing late radiation morbidity, and that carcinoma of the cervix is a good model for testing radiobiologic principles in the clinic.