Methylated DNA-binding protein 2 antisense inhibitors suppress tumourigenesis of human cancer cell lines in vitro and in vivo
Authors: Paul M Campbell
Moshe Szyf
Veronica Bovenzi
Journal: Carcinogenesis (2003)
Discipline: CLINICAL MEDICINE
Keywords: MBD2; DNA demethylase; tumourigenesis; cancer growth; pharmacological inhibition
Affiliations: Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec H3G1Y6, Canada.

Abstract:  Methylated DNA-binding protein 2 (MBD2) has been proposed to function both as a silencer of methylated genes and as a DNA demethylase. Our previous data indicated that knockdown of MBD2 inhibited tumourigenesis of human cancer lines and MBD2-deficient mice were recently shown to be resistant to intestinal tumourigenesis. MBD2 is an attractive anticancer target since MBD2-deficient mice were previously shown to be viable and fertile and knockdown of MBD2 was reported to have no effect on cellular growth parameters of non-transformed cells. In this paper we test the hypothesis that pharmacological inhibition of MBD2 inhibits cancer growth in vivo using human tumour lines implanted in mice as a model. We develop sequence-specific antisense inhibitors of MBD2 and we show that these agents inhibit anchorage-independent growth of human lung (A549) and colorectal (HCT116) cancer cell lines in vitro and tumourigenic growth of human cancer cell xenografts in vivo. MBD2 antisense oligonucleotide does not inhibit the growth of normal and transformed cell lines and does not alter cell cycle parameters in vitro and does not exhibit overt toxicity in vivo in comparison with a scrambled control oligonucleotide, as determined by measuring body mass, blood cell parameters and liver and kidney enzymes. Our data provide a proof of principle that MBD2 is a new anticancer target and that pharmacological inhibition of MBD2 by agents such as the antisense inhibitors described in this paper is a potential new anticancer therapy, which in contrast to the vast majority of current approaches does not target normal progression of the cell cycle. 

Retracted: No

PubPeer comments:
 Figure 1D. Much more similar than expected. ![file] , 
 #1 It came to our attention that a comment was deposited on PubPeer regarding a u03b2-actin loading control in a western blot: Figure 1D published in *Carcinogenesis* 2004: __25__:499-507. We are unaware of any errors made in the assembly of the figure, either at the time of manuscript acceptance approximately 18 years ago or at any time since. We have mobilized strong evidence against the suggestion of inappropriately similar lanes. The comment is suggesting that Lanes 2 and 3 of the u03b2-actin protein blot are more similar than expected, compared to Lanes 5 and 6. It appears that the person who provided the comment was unaware of the fact that this was an actin loading control western blot. The bands look similar because they are supposed to look similar; they are equally quantified (to hundredths of a u00b5g) lanes of a loading control hybridized with an actin antibody. It is expected that all the control bands on the gel will look somewhat alike, especially since the gel was fractionated in the same physical equipment using equal current across the field. If performed well, such an experiment should yield highly similar lanes. The actin antibody is extremely specific, and the actin band is extremely sharp and no size fluctuations are expected between protein samples. Thus, it is _highly likely_ that equally loaded lanes hybridized to actin antibody will look similar, but not identical. Nevertheless, we have enlarged the TIFF image originally submitted to Carcinogenesis to demonstrate, at the pixel level, that Lane 3 and Lane 6 (and similarly, Lanes 2 and 5) are independent (please see Appendix 2, best viewed at 100-300% for details). Portions of Lanes 3 and 6 under the __green bars__ (added for guidance, Appendix 2) are clearly not identical; we can see pixels in the left and right u201ctips of the smileu201d in Lane 3 that are not evident in Lane 6. The bottom of the band signal in Lane 6 (flanked by __orange arrows__) is significantly darker than the corresponding row of pixels in Lane 3. At the same time, the portion of the signal immediately beneath the __blue bar__ in Lane 6 is lighter compared to the corresponding sections of most, but not all of Lane 3 (almost non-existent in 6 and yet light to mid-gray in 3, between __purple asterisks__). Finally, the horizontal density patterns through the middle of Lanes 3 and 6 do not match, with a *pair* of dominantly dark rows in Lane 3, but only a *single* such dark band in Lane 6. In a similar fashion, it can clearly been observed that Lanes 2 and 5 are not replicates of one another. The patterns and densities of horizontal regions indicated by the __black arrows__ and the contiguous vertical columns immediately adjacent to same are different, as are the horizontal densities along the bottoms of each band (between the __fuchsia arrows__): Lane 2 has significantly, but variably, less density than the bottom of Lane 5. In addition to the qualitative images demonstrating that the u03b2-actin distributions and densities are unique and independent as intended, we have bolstered these images with quantification thereof. The details are in Appendix 3 (procedure details in Appendix 6), but in summary, sampling vertically through Lanes 2 and 5 (averaged across adjacent y-axis paths) shows distinct and independent densities and banding, where Lane 2 varies between less than 40% to greater than 280% of matched Lane 5. Similarly, in the u201cbowlu201d or middle of the smile, Lane 2 density varies in a non-linear fashion from less than 35% to 100% (the latter is only 2 of 25 sampled matched areas) compared to respective Lane 5, and horizontally across the bottom of the bands, Lane 5 can be as much as two and a half times (but again, not uniformly) more actin than Lane 2 (Horizontal Bottom in Appendix 3, Page 1). In the evaluation for Lanes 3 and 6, we compared fifteen (3 vertical columns of 5 contiguous rows) paired samples (green bars in Appendix 2) within the u201ctips of the smileu201d for each of the left and right sides (Appendix 3, Page 2). The density analysis shows that there is significant and non-uniform variability in these regions: Lane 3 is between 45% and 333% or 350% (left tip and right tip, respectively) as dense as matched areas of Lane 6. Up to 80% of the paired samples fall outside of a rather generous 90%-110% window of identity. Again, density analysis shows variability vertically through the u201cwaistu201d of Lanes 3 and 6 (corresponding to the blue bar in the grayscale images of Appendix 2). In the quantification, the Lane 3 dual peak density is evident compared to the sharper singular peak signal of Lane 6 previously discussed above. Lane 3 density shifts non-linearly from less than 30% to 213% of Lane 6. Similarly, the bottoms of the bands show negligible density numbers for Lane 3 that are as little as 22% signal of corresponding Lane 6 matched pixels, again with non-uniform differences horizontally. In summary, in grayscale both qualitatively and quantitatively, the loading control bands in question are mostly (up to 80%) dissimilar, indicating that the lanes are independent as designed and expected. ____ Additionally, we analyzed merged overlays of the bands in question (Appendix 4), with one set of lanes assigned red and the other green, and then the ImageJ merged images compared and quantified for similarity or differences between red, green, and yellow saturation (methodology in Appendix 6). At first glance, the images may be perceived as fairly similar, with some differences. The human eye responds most sensitively to light around 550 nm in wavelength due to an overlap of M and L cone receptors(1), and this aligns with yellow-green. Additionally, the human eye responds more strongly to brightness, and since pure yellow has a relative perceived lightness of 94% (while green and red are 80% and 54%, respectively(2)), the eye is disproportionately drawn to and the visual cortex over-values the amount of yellow compared to red or green. When hue and lightness were confounded, participants with normal (or corrected-to-normal) vision were able to most strongly visually identify subfigures due to differences in lightness alone(3), and again, yellow has the greatest perceived lightness of all non-white colors(4). People are somewhat limited in how much color information they can engage, remember, and report.(5) Humans perceive that there is more yellow (i.e. identical overlay) than there actually is because our vision and cortex overestimate it due to brighter color, higher contrast, and heightened spectral response. However, as noted by our *quantification* of the bands (Appendix 5), in reality, much of the merge shows significant regions of unmatched (i.e. more red than green, and *vice versa*, and even areas where the predominant channel switches and then switches back) compared to pairs that are true yellow (i.e. identical). They demonstrate across both pairs of bands in question, across multiple contiguous sampled regions per merged pair, more than 75% (orange cells in Appendix 5) of the merge doesnu2019t even fall within the rather generous 20 point window surrounding identity, and that metric of difference rises to ~83% (in line, numerically, with the dissimilarity shown in the grayscale quantification above) when we apply a more reasonable 95%-105% bracket of true similarity (again, the u201cmost yellowu201d reference sample shows a 99.5% *actual* red green match). The numbers are frank, and they are not restricted to a single sample or region or pair of lanes. If the bands here were duplicated as the PubPeer comment implies, there would be negligible differences for each and every portion of the bands, and that is clearly not the case as shown by the merged images. Parts of the merge are more red than yellow, while other sections are more green than yellow, and these differences are both qualitatively and quantitatively stochastic. This decidedly shows that the bands are similar as expected, but independent. If the bands were duplicates, they would show exactly the same gray densities in the pixels (they do not) and exactly the same intensity of yellow saturation and hue, without red or green dominance (they do not). The numbers clearly and quantitatively disagree with u201cmore similar than expectedu201d. More than three-quarters of the band overlay isnu2019t even close to being similar. We illustrate qualitatively and numerically that the u03b2-actin bands are significantly different in density and banding patterns, at the edges, in the middle, across multiple regions and segments. In our diligence and transparency, we submitted these analyses to the Editor of *Carcinogenesis*. His reply, as we would have expected, confirms our findings as published and refutes any suggestion of figure mismanagement: ___ Tue 9 28 2021 12:37 PM Dear Dr Campbell Thank you very much for your e-mail. Your explanation is acceptable, and the journal will not need to take any further action, especially as you are submitting your additional analysis to PubPeer. Sincerely Dr Curtis C Harris Editor-in-Chief Carcinogenesis: Integrative Cancer Research carcinogenesis.editorialoffice@oup.com ___ Respectfully, ttttttt Paul Campbell, Ph.D. Moshe Szyf, Ph.D. _____ 1.tAbramov I and Gordon J. Color appearance: on seeing red--or yellow, or green, or blue. Annu Rev Psychol. 1994;45:451-85 2.thttp: www.workwithcolor.com color-luminance-2233.htm 3.tStuart et al. The role of lightness, hue and saturation in feature-based visual attention. Vision Res 2014;96:25-32 4. thttp: changingminds.org explanations perception visual light_attracts_eye.htm 5.tCohen MA et al. The limits of color awareness during active, real-world vision. Proc Natl Acad Sci USA. 2020;117:13821-13827 ![file] ![file] ![file] ![file] ![file] ![file] ![file] ,