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  10 Jun 2003 Version: Date: 
 julio Aguirre-Ghiso Reviewer: 
 A paper whose findings are important to those with closely related research Level of interest: 
interests 
 Accept after discretionary revisions Advice on publication: 
This report provides evidence indicating that activation of the Mek-Erk pathway in hepatocellular 
carcinoma is a critical determinant in the proliferative capacity of these tumors. The mechanism proposed involves the activation by MEK-ERK signaling of a survival pathway that prevents 
cytochrome C release, cleavage of caspases 3 and 7 and PARP cleavage. This work proceeds from 
convincing data showing that in human tumor samples there is increased phosphorylation of Mek1 
and this correlates, although not completely, with increased phsphorylation of ERK1/2. Moreover, ERK protein appears to be increased in tumor vs. normal tissue samples. Using genetic (active mutant of Mek) and pharmacological approaches, the authors provide functional data showing that 
the Mek-Erk pathway is important for the survival and proliferation of HepG2 cells in vitro and in 
vivo. Taken together, the results presented by Huynh et al. provide relevant information about the role of this pathway in regulating hepatocarcinoma proliferation. 
Overall this manuscript is coherent, the hypothesis is clear and the experiments support the authors? 
conclusions. However, in order to become acceptable some revisions should be considered. 
Compulsory revisions: 
Treatment of HepG2 cells with UO126 in vitro causes a concomitant reduction in BrdU incorporation 
and an increase in apoptosis. However, the authors attribute the increased proliferation of 
HepG2-Mek cells in vivo only to a reduction from 6% to 1% in apoptosis in vector vs. Mek expressing 
cells, respectively. As pointed by the authors, in vitro conditions differ from the in vivo situation, thus 
the induction of apoptosis observed in vitro may not be as dramatic in vivo and in fact an increase in 
mitogenesis and not in apoptosis may explain this result. BrdU incorporation in vivo or staining for PCNA or Ki67 should be performed on tumor sections of HepG2-vector and HepG2-Mek tumors to distinguish between these two possibilities. 
Tumors produced by vector and Mek expressing cells, appear both to grow very slowly for ~2 months 
and then Mek expressing tumors grow faster. How do the authors reconcile this long latency period 
with the fact that these tumors express an active Mek? 
Discretionary revisions: 
Table I could be removed since the data is clearly summarized in the text. 
Fig 1 and 2: the authors claim that there is more total ERK1/2 protein. Were the MAPK levels 
normalized to tubulin levels? It appears that in T there is more tubulin than in N (clearly seen in A and C), thus it is possible that it is a loading problem and not an actual increase in Erk (MAPK) 
protein. Also the P-ERK levels appear to accompany the increase in ERK protein. A ratio of 
P-ERK/ERK should be calculated by densitometry to estimate the specific increase in ERK phosphorylation when comparing N and T samples. ERK2 phosphorylation and in some cases the protein appears to change even more dramatically than ERK1. Is there a possible interpretation of 
these changes? 
Fig 1, 2 and 3: units appear not to be in percent (%) of control. 
Fig 3: it is not clear if the levels of P-MEK are normalized to total MEK protein. P-Mek/Mek ratio. 
Fig7 is not very convincing, maybe a larger magnification may illustrate better the authors? 
description. 
Fig 10: Is there an explanation for the very low basal levels of ERK-phosphorylation in HepG2 cells 
and the weak induction that active Mek induces? Could this explain the slow proliferation in vivo? 
The statement indicating that Mek alone may be responsible for protecting these cells from 
apoptosis should be reconsidered. Although Akt appears not to play a role in this process, because tumor cells in vivo interact with the extracellular matrix, survival pathways activated by for example integrin-activated FAK signaling (Almeida et al., J Cell Biol. 2000 May 1;149(3):741-54) may be 
important in suppressing apoptosis. 
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  10 Jun 2003 Version: Date: 
 julio Aguirre-Ghiso Reviewer: 
 A paper whose findings are important to those with closely related research Level of interest: 
interests 
 Accept after discretionary revisions Advice on publication: 
This report provides evidence indicating that activation of the Mek-Erk pathway in hepatocellular 
carcinoma is a critical determinant in the proliferative capacity of these tumors. The mechanism proposed involves the activation by MEK-ERK signaling of a survival pathway that prevents 
cytochrome C release, cleavage of caspases 3 and 7 and PARP cleavage. This work proceeds from 
convincing data showing that in human tumor samples there is increased phosphorylation of Mek1 
and this correlates, although not completely, with increased phsphorylation of ERK1/2. Moreover, ERK protein appears to be increased in tumor vs. normal tissue samples. Using genetic (active mutant of Mek) and pharmacological approaches, the authors provide functional data showing that 
the Mek-Erk pathway is important for the survival and proliferation of HepG2 cells in vitro and in 
vivo. Taken together, the results presented by Huynh et al. provide relevant information about the role of this pathway in regulating hepatocarcinoma proliferation. 
Overall this manuscript is coherent, the hypothesis is clear and the experiments support the authors? 
conclusions. However, in order to become acceptable some revisions should be considered. 
Compulsory revisions: 
Treatment of HepG2 cells with UO126 in vitro causes a concomitant reduction in BrdU incorporation 
and an increase in apoptosis. However, the authors attribute the increased proliferation of 
HepG2-Mek cells in vivo only to a reduction from 6% to 1% in apoptosis in vector vs. Mek expressing 
cells, respectively. As pointed by the authors, in vitro conditions differ from the in vivo situation, thus 
the induction of apoptosis observed in vitro may not be as dramatic in vivo and in fact an increase in 
mitogenesis and not in apoptosis may explain this result. BrdU incorporation in vivo or staining for PCNA or Ki67 should be performed on tumor sections of HepG2-vector and HepG2-Mek tumors to distinguish between these two possibilities. 
Tumors produced by vector and Mek expressing cells, appear both to grow very slowly for ~2 months 
and then Mek expressing tumors grow faster. How do the authors reconcile this long latency period 
with the fact that these tumors express an active Mek? 
Discretionary revisions: 
Table I could be removed since the data is clearly summarized in the text. 
Fig 1 and 2: the authors claim that there is more total ERK1/2 protein. Were the MAPK levels 
normalized to tubulin levels? It appears that in T there is more tubulin than in N (clearly seen in A and C), thus it is possible that it is a loading problem and not an actual increase in Erk (MAPK) 
protein. Also the P-ERK levels appear to accompany the increase in ERK protein. A ratio of 
P-ERK/ERK should be calculated by densitometry to estimate the specific increase in ERK phosphorylation when comparing N and T samples. ERK2 phosphorylation and in some cases the protein appears to change even more dramatically than ERK1. Is there a possible interpretation of 
these changes? 
Fig 1, 2 and 3: units appear not to be in percent (%) of control. 
Fig 3: it is not clear if the levels of P-MEK are normalized to total MEK protein. P-Mek/Mek ratio. 
Fig7 is not very convincing, maybe a larger magnification may illustrate better the authors? 
description. 
Fig 10: Is there an explanation for the very low basal levels of ERK-phosphorylation in HepG2 cells 
and the weak induction that active Mek induces? Could this explain the slow proliferation in vivo? 
The statement indicating that Mek alone may be responsible for protecting these cells from 
apoptosis should be reconsidered. Although Akt appears not to play a role in this process, because tumor cells in vivo interact with the extracellular matrix, survival pathways activated by for example integrin-activated FAK signaling (Almeida et al., J Cell Biol. 2000 May 1;149(3):741-54) may be 
important in suppressing apoptosis. 
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  10 Jun 2003 Version: Date: 
 julio Aguirre-Ghiso Reviewer: 
 A paper whose findings are important to those with closely related research Level of interest: 
interests 
 Accept after discretionary revisions Advice on publication: 
This report provides evidence indicating that activation of the Mek-Erk pathway in hepatocellular 
carcinoma is a critical determinant in the proliferative capacity of these tumors. The mechanism proposed involves the activation by MEK-ERK signaling of a survival pathway that prevents 
cytochrome C release, cleavage of caspases 3 and 7 and PARP cleavage. This work proceeds from 
convincing data showing that in human tumor samples there is increased phosphorylation of Mek1 
and this correlates, although not completely, with increased phsphorylation of ERK1/2. Moreover, ERK protein appears to be increased in tumor vs. normal tissue samples. Using genetic (active mutant of Mek) and pharmacological approaches, the authors provide functional data showing that 
the Mek-Erk pathway is important for the survival and proliferation of HepG2 cells in vitro and in 
vivo. Taken together, the results presented by Huynh et al. provide relevant information about the role of this pathway in regulating hepatocarcinoma proliferation. 
Overall this manuscript is coherent, the hypothesis is clear and the experiments support the authors? 
conclusions. However, in order to become acceptable some revisions should be considered. 
Compulsory revisions: 
Treatment of HepG2 cells with UO126 in vitro causes a concomitant reduction in BrdU incorporation 
and an increase in apoptosis. However, the authors attribute the increased proliferation of 
HepG2-Mek cells in vivo only to a reduction from 6% to 1% in apoptosis in vector vs. Mek expressing 
cells, respectively. As pointed by the authors, in vitro conditions differ from the in vivo situation, thus 
the induction of apoptosis observed in vitro may not be as dramatic in vivo and in fact an increase in 
mitogenesis and not in apoptosis may explain this result. BrdU incorporation in vivo or staining for PCNA or Ki67 should be performed on tumor sections of HepG2-vector and HepG2-Mek tumors to distinguish between these two possibilities. 
Tumors produced by vector and Mek expressing cells, appear both to grow very slowly for ~2 months 
and then Mek expressing tumors grow faster. How do the authors reconcile this long latency period 
with the fact that these tumors express an active Mek? 
Discretionary revisions: 
Table I could be removed since the data is clearly summarized in the text. 
Fig 1 and 2: the authors claim that there is more total ERK1/2 protein. Were the MAPK levels 
normalized to tubulin levels? It appears that in T there is more tubulin than in N (clearly seen in A and C), thus it is possible that it is a loading problem and not an actual increase in Erk (MAPK) 
protein. Also the P-ERK levels appear to accompany the increase in ERK protein. A ratio of 
P-ERK/ERK should be calculated by densitometry to estimate the specific increase in ERK phosphorylation when comparing N and T samples. ERK2 phosphorylation and in some cases the protein appears to change even more dramatically than ERK1. Is there a possible interpretation of 
these changes? 
Fig 1, 2 and 3: units appear not to be in percent (%) of control. 
Fig 3: it is not clear if the levels of P-MEK are normalized to total MEK protein. P-Mek/Mek ratio. 
Fig7 is not very convincing, maybe a larger magnification may illustrate better the authors? 
description. 
Fig 10: Is there an explanation for the very low basal levels of ERK-phosphorylation in HepG2 cells 
and the weak induction that active Mek induces? Could this explain the slow proliferation in vivo? 
The statement indicating that Mek alone may be responsible for protecting these cells from 
apoptosis should be reconsidered. Although Akt appears not to play a role in this process, because tumor cells in vivo interact with the extracellular matrix, survival pathways activated by for example integrin-activated FAK signaling (Almeida et al., J Cell Biol. 2000 May 1;149(3):741-54) may be 
important in suppressing apoptosis. 
Competing interests: 
None declared. 
assistant