Download REVIEW Title: p53 expression in human colon cancer tumors in

REVIEW
Title: p53 expression in human colon cancer tumors in nude mice after siRNA CD44
gene therapy
In general this study was well written and only minor revisions to the introduction and
discussion are suggested. However major revisions to the experimental design are
required. The following comments serve to enhance the readability, clarify certain
methods, and strengthen the experimental design and conclusions. A clearer justification
as to how this study adds to the existing knowledge base (i.e. the Subramaniam et al.,
2007 paper) should be included.
Specific Comments:
1. The manuscript should be formatted as one column (not two)
2. References should appear as numbers throughout the manuscript, then listed in the
order they appear in the text after the discussion section. Please use a program
like endnote or refman.
3. Figures should appear after the reference section (not within the body of the
manuscript)
Abstract
1. Line 5: it appears a word is missing. Consider adding “cancer” or “tumor”
following the word “colon”.
2. This sentence should be reworded (perhaps into 2 sentences): “Using this
xenograft mouse model, which had a reduced CD44 expression after siRNA
CD44 gene therapy, we were able to demonstrate a significant decrease in p53
expression that is associated with the inhibition of CD44 expression in tumors
derived from human colon cancer cells”.
3. Please clarify whether you mean an increase or decrease in p53 expression when
you say “mutations” in the sentence “Mutations in the p53 gene result in
decreased genetic stability, reduced apoptosis, and increased survival and
propagation of cells with damaged DNA”
Introduction
1. References should be cited whenever a statement of fact is made, not just at the
end of each paragraph. This applies to much of the first paragraph of the
introduction and should be considered throughout the manuscript.
2. I suggest changing the term “colonic homeostasis” to “epithelial cell turn over in
the colon” in the sentence “Colonic homeostasis involves a 6-day migration of the
cells…”.
3. Consider making the edit: “In normal colonic epithelium, CD44 is expressed in
dividing cells at the base of the crypt instead of but not the non-proliferating
epithelium of the upper crypt and luminal surface”
4. The term “association” and “modulation” are vague in the sentence: “CD44 is
also found to be associated with the characteristic tumor stem cell phenotype and
the modulation of the directional motility and migration of human colon cancer
cells.” Consider using specific terms, such as “increase” or “decrease”, etc.
5. Consider making the edit “Recently, it has been suggested that CD44 provides
resistance to apoptosis using in human colon tumors xenotrasplanted in mice after
siRNA mediated CD44 knock down.” Also, the clarity of the introduction would
be improved if it was stated that the knock down was specifically in the human
cells not the animal (mouse cells).
6. Please state “RNA inhibition (RNAi)” before using the term RNAi.
7. Start a new paragraph and consider rewording: “Previous Intratumoral RNAi of
CD44 work using gene therapy with siRNA CD44 plasmid complex resulted in
has been shown to suppress tumor growth suppression and increased apoptosis in
nude mice (add Subramanian et al., 2007 reference here but remove it from the
sentence following this one in the text).
8. Consider rewording: “The p53 gene is located on the short arm of chromosome
17, and its protein product acts as a transcription factor to maintain genetic
integrity. It does so through induction by arresting cell cycle arrest at the G1-S
transition for DNA repair and induction of apoptosis, for resulting in tumor
growth suppression in stressed cells”
Methodology
1. Consider adding “were” to “…cancer cell line, were xenotransplanted…”
2. Section “siRNA CD44 gene therapy tumor samples”: it is necessary that the
experimental design is written out, at least in brief, and not simply referred to in
another manuscript. The general sequence of events needs to be included. For
example, it isn’t clear whether the human cells were silenced before or after being
placed in the mice. This is especially important since the reference cited used
many different approaches in their methods and it is not clear which samples were
used in THIS study. Don’t forget to describe the control group.
3. What is the sample size for the experimental and control groups?
4. The “antibody” information should be incorporated into the section describing
immunohistochemistry. This was well written.
5. Start a new paragraph and consider rewording: “Each slide was first scanned with
a at low magnification, and Three 3 tumor fields at 20X magnification were per
slide were selected for analysis of p53 expression for observation and
measurements power.” Also, consider clarifying whether each slide represents 1
tumor from 1 animal. How were these sections chosen?
6. How were the cells counted? By eye or using software?
7. Were all the cells in the sections tumor cells? If not, how did you ensure all the
cells that were counted were human tumor cells?
8. Why was the percentage number/um2)used? Do you mean # positive cells/area?
If so, this is not appropriate as there may have been a difference in total cells
(stained and unstained) in the 2 experimental groups due to differences in
apoptosis? Shouldn’t the proportion be total # immunostained nuclei/total nuclei?
Why use per unit area?
Results
1. Consider rewording: “We tested measured the expression of p53 protein in the our
xenograft mouse model and found that nuclear immunostaining for p53 was found
to be significantly reduced by 16% in the tumors that had received the siRNA
CD44 gene therapy, when compared to the mock transfected vector control group,
respectively (Figure 11A, Figure 1B). The reduction of p53 expression in siRNA
CD44 gene therapy treatment group was significant, when compared to the mock
transfectant vector control group (Figure 2, p<0.05).”
2. If the p-value is greater that 0.001 it should be specified (don’t use p<0.05).
3. In your introduction you state “…while the mutant p53 has a prolonged half-life
and accumulates in the nucleus, where it is detectable by immunohistochemistry”.
Please clarify the following points:
a. p53 is a tumor suppressor gene, therefore elevations in wildtype p53
expression should be related to reduced cancer activity.
b. Wont the primary antibody you used stain for wildtype p53? If so,
shouldn’t p53 expression be increased in CD44-silenced tunors?
c. Are you assuming the reduction in p53 expression observed in your
experiment is a reduction in mutant p53? And if so, please clarify how
you justify making that assumption?
Discussion
1. Consider rewording: “Earlier studies from our laboratory using various CD44
models led us to conclude suggest that…”
2. Add a reference to “HT-29 cells express one of the highest levels of CD44 among
human colon cancer cell lines”
3. It is unclear whether the following sentences are referring to the current
manuscript or the study previously performed by the lab:
a. “In this study, intratumoral delivery of siRNA CD44 was accomplished
using polyethyleneimine (PEI).”
b. “Using this mouse xenograft model, we demonstrate in this study that a
significant decrease in the frequency of p53 expression is associated with
a reduction of CD44 after siRNA CD44 gene therapy, consistent with the
therapeutic pressure that was used.”
4. “The present study has undertaken to examine one of these questions: the specific
role of p53 in relation to CD44 in the context of apoptosis.” The “specific role” is
not what the study investigated. This needs to be reworded.
5. Consider rewording: : We exploited our previous observation that induced
reduction of CD44 expression by siRNA CD44 gene therapy results in increased
apoptosis”
References
1. Roughly 65 references are listed in the reference section. This is too many.
Please limit the references list to references that are cited in the manuscript.
Please limit the number of references cited in the text.
Figures
1. Please include whether the Figure 2 is showing mean +/- SD.