Paul V. Newcomb

Insulin-like Growth Factor-binding Protein (IGFBP-3) Predisposes Breast Cancer Cells to Programmed Cell Death in a Non-IGF-dependent Manner

Insulin-like growth factor (IGF) -independent growth inhibition of human breast cancer cells, Hs578T, by IGF-binding protein-3 (IGFBP-3) has previously been demonstrated. Cell growth is a balance between proliferation and programmed cell death (apoptosis). We have investigated whether IGFBP-3 can affect apoptosis of Hs578T cells. As no induction of apoptosis was found, we also investigated its effect on the response to ceramide, an intracellular second messenger that mediates the signal for apoptosis. Using the cell permeable ceramide analogue, C2, induction of apoptosis was established by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay, trypan blue uptake, morphological criteria, and flow cytometry. Incubation of cells with non-glycosylated IGFBP-3 (ngIGFBP-3; 0.5–100 ng/ml) resulted in no growth inhibition or increase in apoptosis; whereas, C2 (1–30 μm) resulted in a dose-dependent induction of apoptosis. Addition of IGFs to the cells, alone or with C2, elicited no response in terms of proliferation or survival, respectively. When the cells were preincubated with ngIGFBP-3 before addition of C2 (2–5 μm), apoptosis was accentuated in a dose-dependent manner (at 100 ng/ml IGFBP-3, apoptosis increased from 11 to 88%). In conclusion, we found that IGFBP-3 had no direct inhibitory effect on Hs578T cells but could accentuate apoptosis induced by the physiological trigger ceramide in an IGF-independent manner.

Differential IGF‐independent effects of insulin‐like growth factor binding proteins (1–6) on apoptosis of breast epithelial cells

We have demonstrated previously that insulin‐like growth factor binding protein (IGFBP)‐3 alone has little growth inhibitory effect on Hs578T human breast cancer cells, but that it can dramatically accentuate the apoptotic response to the physiological trigger, ceramide, in an IGF‐independent manner. We have now studied the potential of other IGFBPs (1–6) to interact with apoptotic signalling pathways. Hs578T cells were preincubated with a binding protein (100 ng/ml) for 24 h, followed by co‐incubation of the binding protein with an apoptotic dose of ceramide or RGD‐containing peptide for a further 24 h. Apoptosis was assessed using flow cytometry, MTT (3‐[4,5‐Dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide; thiazolyl blue) assay and morphological assessment. Binding protein profiles were determined using ligand and immunoblotting techniques. Each of the IGFBPs (1–6) alone had no significant (P > 0.05) growth inhibitory effects relative to control cells. In contrast to IGFBP‐3, which significantly (P < 0.05) accentuated C2‐induced apoptosis, IGFBP‐1, ‐2, and ‐6 had no effect, whereas IGFBP‐4 and ‐5 each caused marked (P < 0.01) inhibition of ceramide‐induced programmed cell death. Apoptosis induced by RGD was also significantly (P < 0.05) reduced by IGFBP‐5, whereas IGFBP‐3 had no effect. These data provide evidence to suggest that individual IGFBPs have specific IGF‐independent effects and act differentially on apoptotic signalling pathways. J. Cell. Biochem. 75:652–664, 1999.

Multifaceted roles of TNF-α in myoblast destruction: A multitude of signal transduction pathways

In catabolic conditions, such as cancer cachexia, a balance favouring a cytokine environment culminates in muscle destruction. Utilising an in vitro model to mimic muscle wasting, we elucidate here the multifaceted roles that one such cytokine, TNF‐α, invokes in the degeneration process. Treatment of C2 skeletal myoblasts with TNF‐α not only suppresses morphological and biochemical differentiation, but following an initial wave of proliferation, and of survival (24 h), induces apoptosis. Investigating the mechanisms underlying these diverse actions of TNF‐α, we demonstrate that cell replication is dependent on rapid and sustained activation of MAP kinase. Map kinase is not, however, central to the death process, which is associated with a progressive rise in caspase‐8 activity, and is accompanied by sustained activation of JNK1 and transient activation of JNK2. Caspase inhibition caused a dose responsive reduction in cell death, while inhibition of the JNKs caused a significant increase in apoptosis. We further report that PI3 kinase is not involved in conferring early protection against TNF‐α‐induced death. By contrast, inhibition of NF‐κB in the presence of TNF‐α culminates in increased cell cycle progression, decreased gadd45β expression and significant and precociously increased cell death, when compared with TNF‐α alone. Our results begin to characterise the mechanisms underlying the acute mitogenic and anti‐apoptotic roles of TNF‐α, which appear to be defined by a balance between MAP kinase, Jun kinase (JNK), NF‐κB and gadd45β. They establish that inhibition of any one of these molecules, as may occur following caspase activation, could eliminate vital stem cells required for skeletal muscle regeneration during chronic catabolic conditions. J. Cell. Physiol. 198: 237–247, 2004© 2003 Wiley‐Liss, Inc.

INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3) POTENTIATES PACLITAXEL-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS

Variability in response to chemotherapy is poorly understood. Paclitaxel‐induced apoptosis was assessed in human Hs578T breast cancer cells, using the MTT assay , cell counting, morphological features and flow cytometry. Pre‐dosing cells with non‐glycosylated insulin‐like growth factor binding protein‐3 (ngIGFBP‐3) had no effect on the cells per se but accentuated paclitaxel‐induced apoptosis. The apoptotic pathway was further examined by measuring caspase‐3 activity in cell lysates at time points over 48 hr after dosing with paclitaxel. Activity increased significantly, and Western immunoblots for caspase‐3 in conditioned media showed that the inactive precursor decreased after incubation with paclitaxel. Endogenous production of IGFBP‐3 by the cells after incubation with paclitaxel was evaluated using Western ligand blotting, specific IGFBP‐3 immunoblotting and radioimmunoassay. Paclitaxel increased endogenous IGFBP‐3, which was further increased if the cells had been pre‐dosed with ngIGFBP‐3. These findings suggest that IGFBP‐3 may be an important modulator of paclitaxel‐induced apoptosis. Int. J. Cancer 88:448–453, 2000.

IGFBP-3 PROLONGS THE p53 RESPONSE AND ENHANCES APOPTOSIS FOLLOWING UV IRRADIATION

Neoplastic transformation is characterised by an imbalance in favour of cell growth over programmed cell death (apoptosis). The tumour‐suppressor gene p53, responsible for maintaining cell‐cycle control, is mutated in the majority of human cancers. Loss of function of the target genes of p53 are therefore important in tumourigenesis. One such target gene is the insulin‐like growth factor binding protein‐3 (IGFBP‐3), an extracellular protein responsible for the carriage of IGF‐I but which can act independently of IGF‐I, inhibiting cell growth and enhancing apoptosis. Using the KYSE190 oesophageal carcinoma cell line, we have demonstrated that IGFBP‐3 alone has no effect on cell growth or cell survival. However, it significantly enhanced apoptosis, with a 67% increase in the pre‐G1 peak on flow cytometry following UV irradiation. The increase in p53 was enhanced and prolonged when cells are stressed in the presence of IGFBP‐3. These data suggest an autocrine/paracrine feedback loop exists between IGFBP‐3 and p53, which may provide the social control necessary to maintain normal tissue homeostasis. Int. J. Cancer 88:336–341, 2000.

Human papillomavirus and oesophageal squamous cell carcinoma in the UK

Human papillomavirus (HPV) has previously been identified in up to 67% of squamous cell carcinomas of the oesophagus. In particular, HPV types of 16 and 18 are believed to play an important role in neoplastic transformation, by means of their oncoproteins E6 and E7. Most of these studies, however, pertain to areas of high incidence of squamous cell carcinoma of the oesophagus (the Far East and South Africa). It is not known if HPV plays any role in the development of oesophageal squamous cell carcinoma in the UK, where the tumour is relatively uncommon. The polymerase chain reaction was used to examine frozen tissue from 22 oesophageal squamous cell carcinomas for the presence of specific DNA sequences from oncogenic strains of HPV. PCR products were further analysed by Southern blot hybridization. No HPV sequences were detected in any tumours. These results suggest that these types of HPV are not associated with oesophageal squamous cell carcinoma in this country. It is unlikely, therefore, that HPV plays a significant role in the pathogenesis of squamous cell carcinoma of the oesophagus in the UK.

Prognostic value of TP53 codon 72 polymorphism in advanced gastric adenocarcinoma

Purpose: A common polymorphism of the tumor suppressor gene TP53 at codon 72 has been associated with human cancer susceptibility. The prognostic role of the polymorphism was assessed in 102 patients with advanced gastric adenocarcinoma. Experimental Design: We followed up 102 consecutive Caucasian patients with advanced gastric adenocarcinoma for >5 years and determined the status of the TP53 codon 72 polymorphism in DNA samples extracted from archived gastric tissues. Results: The frequency of the arginine homozygous allele was positively correlated to patient age at baseline (P = 0.002). However, the age-related increase in the percentage of codon 72 arginine p53 was not correlated to the prognosis for gastric cancer patients. Multivariable analysis in patients who had surgery showed that baseline age may be inversely associated with patient survival (odds ratio, 1.1; 95% confidence interval, 1.0–1.2; P = 0.02). Furthermore, alcohol consumption may be associated with reduced survival (P = 0.06). Conclusions: These findings indicate that codon 72 arginine p53 may not be associated with a prolonged survival in patients with advanced gastric adenocarcinoma, but further study is needed to assess whether this polymorphism is associated with a late onset or slow progress of early gastric adenocarcinoma.

An Immunohistochemical and Prognostic Evaluation of Cathepsin D Expression in 105 Bladder Carcinomas

Cathepsin D is a widely expressed aspartyl lysosomal protease. Clinical studies in several tumor types have shown a strong correlation between cathepsin D expression and tumor progression. In breast carcinoma, its expression is an independent prognostic factor associated with an increased risk of death. However, there have been no studies evaluating cathepsin D in bladder tumors. Therefore, the aim of this study was to determine the pattern of expression of cathepsin D in a large series of bladder carcinomas and assess its role as a prognostic factor against established variables. The tumors from 105 patients (median age 73) (median follow-up 26 months) with transitional cell carcinoma of bladder were examined. Forty-nine patients had superficial tumors (16 pTa; 33 pT1), 56 had invasive tumors (14 pT2; 42 pT3); there were 35 grade 1/2 tumors and 70 grade 3 tumors. These were stained by a standard immunohistochemical technique with an anti-cathepsin D monoclonal antibody. All 4 normal bladder specimens were positive for cathepsin D. Fifty-four tumors (51%) were positive for cathepsin D and 51 (49%) were negative. Chi square analysis showed a significant positive relationship between negative cathepsin D expression and stage (p < 0.0005), grade (p < 0.0001) and tumor morphology (p = 0.001). There was no relationship between cathepsin D expression and tumor ploidy (p > 0.1) or patient age (p = 0.09). Univariate analysis of disease-free and overall survival showed that negative cathepsin D expression (p = 0.01 and p = 0.0003 respectively), stage (p = 0.004 and p < 0.005 respectively) and grade (p = 0.02 and p = 0.0007 respectively) were associated with significantly worse prognosis. However, in a multivariate analysis of age, stage, grade and cathepsin D expression, only stage remained significant for overall survival (p < 0.005). The observed result for cathepsin D in the univariate analysis is probably due to its strong association with grade and stage. Nevertheless, cathepsin D status was able to provide additional prognostic information for overall survival in invasive tumors when stratifying for grade (p = 0.047), which suggests that it might provide additional prognostic data within particular tumor stages.

Insulin-like growth factor binding protein-3: relationship to the development of gastric pre-malignancy and gastric adenocarcinoma (United Kingdom).

IGF family proteins play a pivotal role in regulating cell growth and apoptosis in normal and tumour tissues. IGFBP-3 is the major binding protein of IGFs and modulates the bioactivity of IGFs. To examine the role of IGFBP-3 in gastric cancer, an IGFBP3 promoter polymorphism, and serum and gastric mucosal levels of IGFBP-3 were assessed in two independent groups of patients (396 and 117 patients, respectively) with gastroduodenal diseases. There was no significant association between IGFBP-3 polymorphism and different gastroduodenal diseases (p = 0.6), but a significantly higher frequency of CC, a genotype related to lower levels of serum IGFBP-3 previously, were observed in patients with antral intestinal metaplasia when compared with those without this pre-malignancy (p = 0.04). Similarly, data from another independent group of patients further showed that patients with antral or corpus intestinal metaplasia had significantly lower serum levels of IGFBP-3 than those without these changes (p = 0.03 and 0.04, respectively). Furthermore, the percentage of positive IGFBP-3 staining in tumour tissue was significantly higher in patients with well or moderately differentiated tumours than those with poorly differentiated tumours (p = 0.04), indicating that IGFBP-3 may be associated with a better prognosis. In conclusion, our study suggests that IGFBP-3 may be protective against the development of gastric adenocarcinoma by preventing the formation of intestinal metaplasia and improve the prognosis of gastric cancer.

A Comparison Study of Gastric Cancer Risk in Patients with Duodenal and Gastric Ulcer: Roles of Gastric Mucosal Histology and p53 Codon 72 Polymorphism

Gastric ulcer is positively, and duodenal ulcer negatively, associated with the risk of gastric cancer. The relationship between a common p53 polymorphism at codon 72 and gastric cancer risk in patients with gastric and duodenal ulcer was examined in 397 Caucasian patients using PCR-RFLP. Noncardiac cancer patients had a distribution pattern of codon 72 genotypes similar to that of other non-cancer patient groups, though the frequency of the Pro/Pro genotype looks higher in duodenal ulcer. However, patients with cancer of the cardiac region had a significantly higher frequency of the Arg/Arg genotype than patients with chronic gastritis, duodenal ulcer, and noncardiac cancer. There was no significant difference in the distribution patterns between gastric ulcer and noncardiac or cardiac cancer or between gastric and duodenal ulcer. These findings may be a reflection of differences in the interaction between p53 codon 72 polymorphism and local factors in the stomach.