Reviewer's report 
 Over-expression of MEK-MAPK in hepatocellular carcinoma: Its role in tumour progression Title: 
and apoptosis. 
Authors: 
Hung T Huynh ( cmrhth@nccs.com.sg )
Tuyen T.T. Nguyen ( cmrnttt@nccs.com.sg )
Pierce Chow ( gsupc@sgh.com.sg )
Puay Hoon Tan ( gpttph@sgh.com.sg )
Khee Chee Soo ( admskc@nccs.com.sg )
Evelyne Tran ( cmrhth@nccs.com.sg )
 4  20 Jun 2003 Version: Date: 
 Chris Clarke Reviewer: 
 A paper of limited interest Level of interest: 
 Accept after discretionary revisions Advice on publication: 
1. This study provides a detailed analysis of the role of the ras/ERK pathway in hepatocellular 
carcinoma. The authors demonstrate a correlation between the overexpression and activation of 
MEK and ERK and a tumour phenotype. Furthermore, they show that an inhibitor of this pathway induces apoptosis in liver derived cell lines and study molecular events associated with this 
phenomenon. Finally, they demonstrate that although the in vitro growth rate of cells is unaffected by 
MEK over expression, growth as a tumour in immune compromised animals is promoted. The 
experiments that have been performed are appropriate for this study and have been well designed and executed. The major criticism of this work is lack of novelty. 
Compulsory Revisions 
2. Over expression of MEK AND MAPK in hepatocellular carcinoma has already been described 
(Schmidt et al 1997). Although a larger number of tumour samples have been tested by these 
authors, the conclusions that they reach are substantially the same. The authors should refer to the 
previous study. 
3. The concept of inducing apoptosis in cells with an activated ras/MAPK pathway is not new. 
Indeed, another MEK1/2 inhibitor PD98059 was shown to induce apoptosis in the same HepG2 cell 
line used by the authors (Mitsui et al 2001). The work performed in these studies has not been acknowledged by the authors and should be referenced. 
4. PARP was one of the earliest caspase substrates to be identified. However, within the apoptosis 
field, cleavage of PARP is considered to be a late biochemical event during apoptosis, not an early event as the authors assert in the results and discussion the text must be amended. Cleavage of caspase 3, caspase 7 and PARP are still good markers of apoptotic cell death although such 
observations have been made late during necrotic death. 
Discretionary Revisions. 
5. Basing conclusions on a single pharmacological agent can occasionally be misleading. Given the 
availability of other specific MEK inhibitors, the paper would be strengthened if some of the 
experiments, were repeated using another unrelated inhibitor. 
6. An introductory sentence added to the first results paragraph referring to Table1 pathologic data 
would clarify this part of the study. 
Competing interests: 
None declared. 
Reviewer's report 
 Over-expression of MEK-MAPK in hepatocellular carcinoma: Its role in tumour progression Title: 
and apoptosis. 
Authors: 
Hung T Huynh ( cmrhth@nccs.com.sg )
Tuyen T.T. Nguyen ( cmrnttt@nccs.com.sg )
Pierce Chow ( gsupc@sgh.com.sg )
Puay Hoon Tan ( gpttph@sgh.com.sg )
Khee Chee Soo ( admskc@nccs.com.sg )
Evelyne Tran ( cmrhth@nccs.com.sg )
 4  20 Jun 2003 Version: Date: 
 Chris Clarke Reviewer: 
 A paper of limited interest Level of interest: 
 Accept after discretionary revisions Advice on publication: 
1. This study provides a detailed analysis of the role of the ras/ERK pathway in hepatocellular 
carcinoma. The authors demonstrate a correlation between the overexpression and activation of 
MEK and ERK and a tumour phenotype. Furthermore, they show that an inhibitor of this pathway induces apoptosis in liver derived cell lines and study molecular events associated with this 
phenomenon. Finally, they demonstrate that although the in vitro growth rate of cells is unaffected by 
MEK over expression, growth as a tumour in immune compromised animals is promoted. The 
experiments that have been performed are appropriate for this study and have been well designed and executed. The major criticism of this work is lack of novelty. 
Compulsory Revisions 
2. Over expression of MEK AND MAPK in hepatocellular carcinoma has already been described 
(Schmidt et al 1997). Although a larger number of tumour samples have been tested by these 
authors, the conclusions that they reach are substantially the same. The authors should refer to the 
previous study. 
3. The concept of inducing apoptosis in cells with an activated ras/MAPK pathway is not new. 
Indeed, another MEK1/2 inhibitor PD98059 was shown to induce apoptosis in the same HepG2 cell 
line used by the authors (Mitsui et al 2001). The work performed in these studies has not been acknowledged by the authors and should be referenced. 
4. PARP was one of the earliest caspase substrates to be identified. However, within the apoptosis 
field, cleavage of PARP is considered to be a late biochemical event during apoptosis, not an early event as the authors assert in the results and discussion the text must be amended. Cleavage of caspase 3, caspase 7 and PARP are still good markers of apoptotic cell death although such 
observations have been made late during necrotic death. 
Discretionary Revisions. 
5. Basing conclusions on a single pharmacological agent can occasionally be misleading. Given the 
availability of other specific MEK inhibitors, the paper would be strengthened if some of the 
experiments, were repeated using another unrelated inhibitor. 
6. An introductory sentence added to the first results paragraph referring to Table1 pathologic data 
would clarify this part of the study. 
Competing interests: 
None declared. 
Please review this paper.
user
Reviewer's report 
 Over-expression of MEK-MAPK in hepatocellular carcinoma: Its role in tumour progression Title: 
and apoptosis. 
Authors: 
Hung T Huynh ( cmrhth@nccs.com.sg )
Tuyen T.T. Nguyen ( cmrnttt@nccs.com.sg )
Pierce Chow ( gsupc@sgh.com.sg )
Puay Hoon Tan ( gpttph@sgh.com.sg )
Khee Chee Soo ( admskc@nccs.com.sg )
Evelyne Tran ( cmrhth@nccs.com.sg )
 4  20 Jun 2003 Version: Date: 
 Chris Clarke Reviewer: 
 A paper of limited interest Level of interest: 
 Accept after discretionary revisions Advice on publication: 
1. This study provides a detailed analysis of the role of the ras/ERK pathway in hepatocellular 
carcinoma. The authors demonstrate a correlation between the overexpression and activation of 
MEK and ERK and a tumour phenotype. Furthermore, they show that an inhibitor of this pathway induces apoptosis in liver derived cell lines and study molecular events associated with this 
phenomenon. Finally, they demonstrate that although the in vitro growth rate of cells is unaffected by 
MEK over expression, growth as a tumour in immune compromised animals is promoted. The 
experiments that have been performed are appropriate for this study and have been well designed and executed. The major criticism of this work is lack of novelty. 
Compulsory Revisions 
2. Over expression of MEK AND MAPK in hepatocellular carcinoma has already been described 
(Schmidt et al 1997). Although a larger number of tumour samples have been tested by these 
authors, the conclusions that they reach are substantially the same. The authors should refer to the 
previous study. 
3. The concept of inducing apoptosis in cells with an activated ras/MAPK pathway is not new. 
Indeed, another MEK1/2 inhibitor PD98059 was shown to induce apoptosis in the same HepG2 cell 
line used by the authors (Mitsui et al 2001). The work performed in these studies has not been acknowledged by the authors and should be referenced. 
4. PARP was one of the earliest caspase substrates to be identified. However, within the apoptosis 
field, cleavage of PARP is considered to be a late biochemical event during apoptosis, not an early event as the authors assert in the results and discussion the text must be amended. Cleavage of caspase 3, caspase 7 and PARP are still good markers of apoptotic cell death although such 
observations have been made late during necrotic death. 
Discretionary Revisions. 
5. Basing conclusions on a single pharmacological agent can occasionally be misleading. Given the 
availability of other specific MEK inhibitors, the paper would be strengthened if some of the 
experiments, were repeated using another unrelated inhibitor. 
6. An introductory sentence added to the first results paragraph referring to Table1 pathologic data 
would clarify this part of the study. 
Competing interests: 
None declared. 
assistant