Reviewer's report
Title:Truncated LEF-1 is one of the key regulators in the growth of colon
carcinoma
Version: 2Date:6 May 2011
Reviewer: Andrew Quest
Reviewer's report:
Revision of manuscript entitled
“Truncated LEF-1 is one of the key regulators in the growth of colon carcinoma”
by Wang et al
LEF-1 is a transciption factor implicated in the progression of colon cancer. A
truncated form referred to here as LEF-1-dL also exists, which might act as an
inhibitor of LEF-1 function. Alternatively, evidence is available indicating that the
two forms potentially cooperate in the control of cellular function. In colon cancer
cells an increase in the expression of full-length LEF (LEF-1-FL) is observed
while LEF-1-dL presence is reduced. Here the authors first show that alterations
in the presence of these two isoforms are observed in colon samples from
patients. Specifically, increases in LEF-1-dL were detected in normal and
tumor-adjacent tissue while LEF-1-FL prevailed in tumor samples. Subsequently,
the role of these different isoforms was studied in the two colon carcinoma cell
lines HT29 and SW480. Overexpression of LEF-1-dL coincided with reduced
proliferation and increased cell death in vitro. Moreover, expression of LEF-1-dL
reduced migration in response to SDF-1 of HT29 cells in a transwell assay. This
was linked to reduced expression of the chemokine receptor CXCR4. Finally,
tumor growth in vivo, as well as vacularization were reduced in tumors formed by
LEF-1-dL expressing SW480 and HT29 cells. Taken together these results
indicate that variations in the expression of these two LEF-1 variants have a
substantial impact on the behavior of colon cancer cells.
In general the experiments are well done and the results obtained support the
conclusions. However, some issues need to be addressed.
Major comments
In the methods section it is not specifically stated how many patient samples
were obtained and analyzed. Was the total number evaluated 22 as stated in the
results section? Additionally, the authors mention obtaining tumor samples and
adjacent normal tissue. Yet in the results section (see Figure 1) three types of
tissue samples were evaluated, colon carcinomas, adjacent tissue AND normal
tissue. It is not clear from the information available what exactly the samples
analyzed represent. This needs to be clarified.
Moreover, in Figure 1, are the Western blots of the three tissue samples from two
or more patients? Presumably these are representative results. The authors
should indicate in each case, how many they are representative of. The authors
should indicate in all these panels where appropriate molecular weight markers
migrate.
In Figure 2, cells are compared after analysis of the cell cycle by flow cytometry.
Rather surprisingly, no increments in the sub G0/G1 population are detected for
both the SW480-dL and HT29-dL cells. This would be expected based on the
result shown in Figure 3 where an increase in Annexin-V positive cells is
observed for both these cell lines. The authors need to provide an explanation for
this discrepancy and preferably should include the percentage cells in sub G0/G1
in the analysis shown in Figure 2.
In Figure 4, the authors talk about a colony forming assay in panels A-D. In is not
clear from the description provided what exactly the authors are measuring here.
Is this anchorage-independent growth in a colony forming assay? This needs to
be explained better in all relevant sections. In panels B and D, the y-axis should
probably be labeled “Colony numbers” rather than “Clone numbers”.
In Figure 5, the authors analyze expression of their his-tagged constructs in
tumor-derived cells. From the methods section, it is not clear how these cells
were obtained. Moreover, all tagged proteins shown are of the same size.
According to the results in Figure 1, LEF-1-dL is notably smaller. The authors
need to explain better what is being demonstrated. Also molecular weight
markers should be included.
In Figure 6, tumor growth is documented for the different HT29 and SW480
variants. In Figure 7, vascularization of tumors is characterized using different
markers in one set of tumors. However, the information provided is confusing. In
the figure panels, HT29 is mentioned while the figure legend states that tumors
derived from both HT29 and SW480 cells were analyzed. This needs
clarification. Also, the authors should evaluate whether reduced tumor
vascularization observed for cells expressing the LEF-1-dL variant is also
significantly reduced in tumors of the same size. The current analysis detects
differences in tumors of different sizes.
The conclusion at the end of the discussion is that “the balance between the two
forms of LEF-1 might have important consequences for normal growth of colon
cancer cells and cancer”. This statement doesn’t really coincide with the
message provided by the title “Truncated LEF-1 is one key regulador in the
growth of colon carcinoma”. The title needs to be altered to reflect better the
findings of the paper.
The English of the manuscript needs major revision preferably by a native
speaker. There are a number of confusing statements throughout the text. For
instance, at the end of the second paragraph in the results section, the authors
summarize by saying “These results indicate that LEF-1 may play an important
role in colon carcinogenesis”. The statement follows just after a section
describing how HT29 and SW480 cells expressing the different LEF-1 variants
were generated. Thus, it would appear out of place.
Minor comments
Figure 1. should be HeLa rather that Hella.
There are many typo errors throughout the text
Throughout the text several abbreviations are not explained (CSFE, SDF-1,
CXCR4, MMPs, CDK etc). Also is the abbreviation Hif-1a or HIF-1a?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Not suitable for publication unless extensively edited
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Reviewer's report
Title:Truncated LEF-1 is one of the key regulators in the growth of colon
carcinoma
Version: 2Date:6 May 2011
Reviewer: Andrew Quest
Reviewer's report:
Revision of manuscript entitled
“Truncated LEF-1 is one of the key regulators in the growth of colon carcinoma”
by Wang et al
LEF-1 is a transciption factor implicated in the progression of colon cancer. A
truncated form referred to here as LEF-1-dL also exists, which might act as an
inhibitor of LEF-1 function. Alternatively, evidence is available indicating that the
two forms potentially cooperate in the control of cellular function. In colon cancer
cells an increase in the expression of full-length LEF (LEF-1-FL) is observed
while LEF-1-dL presence is reduced. Here the authors first show that alterations
in the presence of these two isoforms are observed in colon samples from
patients. Specifically, increases in LEF-1-dL were detected in normal and
tumor-adjacent tissue while LEF-1-FL prevailed in tumor samples. Subsequently,
the role of these different isoforms was studied in the two colon carcinoma cell
lines HT29 and SW480. Overexpression of LEF-1-dL coincided with reduced
proliferation and increased cell death in vitro. Moreover, expression of LEF-1-dL
reduced migration in response to SDF-1 of HT29 cells in a transwell assay. This
was linked to reduced expression of the chemokine receptor CXCR4. Finally,
tumor growth in vivo, as well as vacularization were reduced in tumors formed by
LEF-1-dL expressing SW480 and HT29 cells. Taken together these results
indicate that variations in the expression of these two LEF-1 variants have a
substantial impact on the behavior of colon cancer cells.
In general the experiments are well done and the results obtained support the
conclusions. However, some issues need to be addressed.
Major comments
In the methods section it is not specifically stated how many patient samples
were obtained and analyzed. Was the total number evaluated 22 as stated in the
results section? Additionally, the authors mention obtaining tumor samples and
adjacent normal tissue. Yet in the results section (see Figure 1) three types of
tissue samples were evaluated, colon carcinomas, adjacent tissue AND normal
tissue. It is not clear from the information available what exactly the samples
analyzed represent. This needs to be clarified.
Moreover, in Figure 1, are the Western blots of the three tissue samples from two
or more patients? Presumably these are representative results. The authors
should indicate in each case, how many they are representative of. The authors
should indicate in all these panels where appropriate molecular weight markers
migrate.
In Figure 2, cells are compared after analysis of the cell cycle by flow cytometry.
Rather surprisingly, no increments in the sub G0/G1 population are detected for
both the SW480-dL and HT29-dL cells. This would be expected based on the
result shown in Figure 3 where an increase in Annexin-V positive cells is
observed for both these cell lines. The authors need to provide an explanation for
this discrepancy and preferably should include the percentage cells in sub G0/G1
in the analysis shown in Figure 2.
In Figure 4, the authors talk about a colony forming assay in panels A-D. In is not
clear from the description provided what exactly the authors are measuring here.
Is this anchorage-independent growth in a colony forming assay? This needs to
be explained better in all relevant sections. In panels B and D, the y-axis should
probably be labeled “Colony numbers” rather than “Clone numbers”.
In Figure 5, the authors analyze expression of their his-tagged constructs in
tumor-derived cells. From the methods section, it is not clear how these cells
were obtained. Moreover, all tagged proteins shown are of the same size.
According to the results in Figure 1, LEF-1-dL is notably smaller. The authors
need to explain better what is being demonstrated. Also molecular weight
markers should be included.
In Figure 6, tumor growth is documented for the different HT29 and SW480
variants. In Figure 7, vascularization of tumors is characterized using different
markers in one set of tumors. However, the information provided is confusing. In
the figure panels, HT29 is mentioned while the figure legend states that tumors
derived from both HT29 and SW480 cells were analyzed. This needs
clarification. Also, the authors should evaluate whether reduced tumor
vascularization observed for cells expressing the LEF-1-dL variant is also
significantly reduced in tumors of the same size. The current analysis detects
differences in tumors of different sizes.
The conclusion at the end of the discussion is that “the balance between the two
forms of LEF-1 might have important consequences for normal growth of colon
cancer cells and cancer”. This statement doesn’t really coincide with the
message provided by the title “Truncated LEF-1 is one key regulador in the
growth of colon carcinoma”. The title needs to be altered to reflect better the
findings of the paper.
The English of the manuscript needs major revision preferably by a native
speaker. There are a number of confusing statements throughout the text. For
instance, at the end of the second paragraph in the results section, the authors
summarize by saying “These results indicate that LEF-1 may play an important
role in colon carcinogenesis”. The statement follows just after a section
describing how HT29 and SW480 cells expressing the different LEF-1 variants
were generated. Thus, it would appear out of place.
Minor comments
Figure 1. should be HeLa rather that Hella.
There are many typo errors throughout the text
Throughout the text several abbreviations are not explained (CSFE, SDF-1,
CXCR4, MMPs, CDK etc). Also is the abbreviation Hif-1a or HIF-1a?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Not suitable for publication unless extensively edited
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
Please review this paper.
user
Reviewer's report
Title:Truncated LEF-1 is one of the key regulators in the growth of colon
carcinoma
Version: 2Date:6 May 2011
Reviewer: Andrew Quest
Reviewer's report:
Revision of manuscript entitled
“Truncated LEF-1 is one of the key regulators in the growth of colon carcinoma”
by Wang et al
LEF-1 is a transciption factor implicated in the progression of colon cancer. A
truncated form referred to here as LEF-1-dL also exists, which might act as an
inhibitor of LEF-1 function. Alternatively, evidence is available indicating that the
two forms potentially cooperate in the control of cellular function. In colon cancer
cells an increase in the expression of full-length LEF (LEF-1-FL) is observed
while LEF-1-dL presence is reduced. Here the authors first show that alterations
in the presence of these two isoforms are observed in colon samples from
patients. Specifically, increases in LEF-1-dL were detected in normal and
tumor-adjacent tissue while LEF-1-FL prevailed in tumor samples. Subsequently,
the role of these different isoforms was studied in the two colon carcinoma cell
lines HT29 and SW480. Overexpression of LEF-1-dL coincided with reduced
proliferation and increased cell death in vitro. Moreover, expression of LEF-1-dL
reduced migration in response to SDF-1 of HT29 cells in a transwell assay. This
was linked to reduced expression of the chemokine receptor CXCR4. Finally,
tumor growth in vivo, as well as vacularization were reduced in tumors formed by
LEF-1-dL expressing SW480 and HT29 cells. Taken together these results
indicate that variations in the expression of these two LEF-1 variants have a
substantial impact on the behavior of colon cancer cells.
In general the experiments are well done and the results obtained support the
conclusions. However, some issues need to be addressed.
Major comments
In the methods section it is not specifically stated how many patient samples
were obtained and analyzed. Was the total number evaluated 22 as stated in the
results section? Additionally, the authors mention obtaining tumor samples and
adjacent normal tissue. Yet in the results section (see Figure 1) three types of
tissue samples were evaluated, colon carcinomas, adjacent tissue AND normal
tissue. It is not clear from the information available what exactly the samples
analyzed represent. This needs to be clarified.
Moreover, in Figure 1, are the Western blots of the three tissue samples from two
or more patients? Presumably these are representative results. The authors
should indicate in each case, how many they are representative of. The authors
should indicate in all these panels where appropriate molecular weight markers
migrate.
In Figure 2, cells are compared after analysis of the cell cycle by flow cytometry.
Rather surprisingly, no increments in the sub G0/G1 population are detected for
both the SW480-dL and HT29-dL cells. This would be expected based on the
result shown in Figure 3 where an increase in Annexin-V positive cells is
observed for both these cell lines. The authors need to provide an explanation for
this discrepancy and preferably should include the percentage cells in sub G0/G1
in the analysis shown in Figure 2.
In Figure 4, the authors talk about a colony forming assay in panels A-D. In is not
clear from the description provided what exactly the authors are measuring here.
Is this anchorage-independent growth in a colony forming assay? This needs to
be explained better in all relevant sections. In panels B and D, the y-axis should
probably be labeled “Colony numbers” rather than “Clone numbers”.
In Figure 5, the authors analyze expression of their his-tagged constructs in
tumor-derived cells. From the methods section, it is not clear how these cells
were obtained. Moreover, all tagged proteins shown are of the same size.
According to the results in Figure 1, LEF-1-dL is notably smaller. The authors
need to explain better what is being demonstrated. Also molecular weight
markers should be included.
In Figure 6, tumor growth is documented for the different HT29 and SW480
variants. In Figure 7, vascularization of tumors is characterized using different
markers in one set of tumors. However, the information provided is confusing. In
the figure panels, HT29 is mentioned while the figure legend states that tumors
derived from both HT29 and SW480 cells were analyzed. This needs
clarification. Also, the authors should evaluate whether reduced tumor
vascularization observed for cells expressing the LEF-1-dL variant is also
significantly reduced in tumors of the same size. The current analysis detects
differences in tumors of different sizes.
The conclusion at the end of the discussion is that “the balance between the two
forms of LEF-1 might have important consequences for normal growth of colon
cancer cells and cancer”. This statement doesn’t really coincide with the
message provided by the title “Truncated LEF-1 is one key regulador in the
growth of colon carcinoma”. The title needs to be altered to reflect better the
findings of the paper.
The English of the manuscript needs major revision preferably by a native
speaker. There are a number of confusing statements throughout the text. For
instance, at the end of the second paragraph in the results section, the authors
summarize by saying “These results indicate that LEF-1 may play an important
role in colon carcinogenesis”. The statement follows just after a section
describing how HT29 and SW480 cells expressing the different LEF-1 variants
were generated. Thus, it would appear out of place.
Minor comments
Figure 1. should be HeLa rather that Hella.
There are many typo errors throughout the text
Throughout the text several abbreviations are not explained (CSFE, SDF-1,
CXCR4, MMPs, CDK etc). Also is the abbreviation Hif-1a or HIF-1a?
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Not suitable for publication unless extensively edited
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests
assistant