Radiosensitization by pan ErbB inhibitor CI-1033 in vitro and in vivo.

PURPOSE: Overexpression of the ErbB family of receptor tyrosine kinases has been associated with uncontrolled growth of many tumor types and, therefore, presents a promising molecular target for cancer therapy. CI-1033 is a small molecule tyrosine kinase inhibitor that differs from other 4-anilinoquinazolines by being a pan ErbB (instead of epidermal growth factor receptor-specific) irreversible (instead of reversible) inhibitor. Therefore, we investigated the antitumor effect of CI-1033 alone and in combination with ionizing radiation in vitro and in vivo. EXPERIMENTAL DESIGN: We selected three human colon carcinoma cell-lines (LoVo, Caco-2, which express activated epidermal growth factor receptor and ErbB-2 family members, and SW620, which does not), and analyzed the effects of CI-1033 both in vitro and in vivo. For in vivo studies LoVo and Caco-2 cells were implanted s.c. in the flank of nude mice. After the tumor reached approximately 100 mm(3), treatment was initiated with 20 mg/kg of CI-1033 (orally once daily x 5 for 3 successive weeks), radiation treatment (a total of 30 Gy given in 2 Gy once daily x 5 for 3 successive weeks), or a combination of both CI-1033 and radiation treatment. RESULTS: We found that exposure of LoVo and Caco-2, but not SW620 cells, to CI-1033 in the range of 1-3 micro M could inhibit constitutive signaling by tyrosine kinases, arrest cell growth, inhibit cells in G(1), stimulate expression of p53, and induce apoptosis. The inhibition of cell growth by CI-1033 seemed to produce only minimal radiosensitization in LoVo and Caco-2 cells. In contrast, the combination of CI-1033 and radiation produced significant (P < 0.0005 and P = 0.0002, respectively) and prolonged suppression of tumor growth in both the tumor types when compared with either treatment alone. CONCLUSIONS: These findings suggest that CI-1033 can increase the effectiveness of radiation therapy. The extent of suppression of tyrosine kinase activity by CI-1033, rather than the amount of activity in untreated cells, seemed to be more closely associated with the efficacy of combination treatment.     

Evaluation of an in vitro model of hepatic inflammatory response by gene expression profiling

The body's response to biochemical stress involves coordinated changes in the expression of several sets of genes that regulate its return to homeostasis. Although several cell culture systems have been utilized for studying such complex physiological events in vitro, their assessment has been limited to biochemical assays on individual genes and proteins, limiting interpretation of the results in a systems context. Advances in genomics provide an opportunity to provide a more comprehensive assessment. In this study, we have used DNA microarrays to profile gene expression dynamics during interleukin 6-stimulated inflammation in hepatocytes maintained in a stable, collagen double-gel in vitro model system. The observed expression profile was also compared with that obtained from rat liver tissue after burn injury to determine the extent and nature of responses captured by the in vitro system. Our results indicate that several aspects of the in vivo hepatic inflammatory response can be captured by the in vitro system at the molecular systems level. Statistical analysis of the mRNA profiles was also used to characterize the temporal response in each model system and demonstrate similar behavior. A small panel of molecules involved in the hepatic acute-phase response was also profiled, using quantitative kinetic polymerase chain reaction, to confirm these observations. These results indicate the utility of the stable hepatocyte culture system for expression profiling of inflammatory states and for providing insights into the interplay of changes in gene expression during complex physiological states.     

Investigational use of ribavirin in the treatment of severe acute respiratory syndrome, Singapore, 2003

OBJECTIVE: Ribavirin is a broad spectrum nucleoside analogue efficacious in the treatment of several viral infections. In the recent severe acute respiratory syndrome (SARS) outbreak, ribavirin was used in various countries against this novel coronavirus. We conducted a retrospective analysis to assess the efficacy of ribavirin in the treatment of SARS in Singapore. METHODS: A total of 229 cases were analysed. Ninety-seven (42.4%) patients received ribavirin at a mean of 6.4 days of illness. Clinical and laboratory parameters taken at the start of ribavirin treatment were compared with day 5, 6 or 7 parameters of those patients not on treatment. The two groups were compared using Fisher's exact test and Student's t-test. Multivariate analysis was performed using Cox regression model with death as the outcome of interest. RESULTS: The treatment group had younger women with more symptoms of myalgia (P < 0.001). The crude death rate was 12.9% in the control and 10.3% (P = 0.679) in the treatment group. After correction for age, male sex, lactate dehydrogenase levels and steroid use, the hazard ratio was 1.03 (95% CI: 0.44-2.41, P = 0.939). CONCLUSION: In this retrospective, uncontrolled cohort analysis, use of ribavirin did not appear to confer any benefit for patients with SARS.     

Activity-based probes for the proteomic profiling of metalloproteases

Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.     

Severe acute respiratory syndrome (SARS) in Singapore: clinical features of index patient and initial contacts

Severe acute respiratory syndrome (SARS) is an emerging viral infectious disease. One of the largest outbreaks of SARS to date began in Singapore in March 2003. We describe the clinical, laboratory, and radiologic features of the index patient and the patient's initial contacts affected with probable SARS.