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. 2012 Jul;39(7):617-24.
doi: 10.1111/j.1346-8138.2012.01553.x. Epub 2012 Apr 9.

Treatment with the cancer drugs decitabine and doxorubicin induces human skin keratinocytes to express Oct4 and the OCT4 regulator mir-145

Sathivel Chinnathambi  1 , Susan Wiechert, Ann Tomanek-Chalkley, Michael C Winter, Jackie R Bickenbach
Affiliations

Treatment with the cancer drugs decitabine and doxorubicin induces human skin keratinocytes to express Oct4 and the OCT4 regulator mir-145

Sathivel Chinnathambi et al. J Dermatol. 2012 Jul.

Abstract

Previously, we showed that transient transfection with OCT4 not only produced high expression of Oct4 in skin keratinocytes, but also caused a generalized demethylation of keratinocyte DNA. We hypothesized that DNA demethylation alone might allow expression of endogenous OCT4. Here, we report that treatment with the cancer drug decitabine results in generalized DNA demethylation in skin keratinocytes, and by 48 h after treatment, 96% of keratinocytes show expression of the endogenous Oct4 protein and the OCT4 repressor mir-145. This is true for keratinocytes only, as skin fibroblasts treated similarly show no OCT4 or mir-145 expression. Decitabine-treated keratinocytes also show increased mir-302c and proliferation similar to other Oct4(+) cells. Treatment with doxorubicin, another cancer drug, induces expression of mir-145 only in cells that already express OCT4, suggesting that Oct4 regulates its own repressor. Co-treatment with decitabine and doxorubicin results first in increased OCT4 and mir-145, then a decrease in both, suggesting that OCT4 and mir-145 regulate each other. The novel strategy presented here provides a regulatable system to produce Oct4(+) cells for transformation studies and provides a unique method to study the effects of endogenous Oct4 in cancer cells and the surrounding somatic cells.

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Figures

Figure 1
Figure 1. Treatment with decitabtine demethylates human skin keratinocyte (HSK) DNA
HSKs were cultured with 25 μM decitiabine for the noted number of days. Treated HSK cultures were normalized to untreated HSKs cultured for the same day. Note, HSKs treated for 24 hours show the least amount of methylation (the highest level of demethylation). n = 3 for each time point.
Figure 2
Figure 2. A single treatment with decitabine is sufficient to allow expression of the endogenous Oct4 protein in HSKs
A) Immunofluorescent image of Oct4 expression in HSKs treated with one dose of 25 μM Decitabine for 48 hours. B) Immunofluorescent image of Oct4 expression in HSKs transfected with pMSCV-OCT4 for 48 hours. Note, transfection hits only ~10% of HSKs, and these cells have extremely high expression of nuclear Oct4. Decitabine treatment affects 96% of HSKs, and these cells show a low expression of Oct4. Blue = DAPI. Red/pink = antibody to Oct4.
Figure 3
Figure 3. A single treatment with decitabine induces expression of endogenous OCT4, mir-145, and mir-302c in HSKs
qPCR data from decitabine-treated HSKs were normalized to untreated HSK controls, which were arbitrarily set at 1.00. Decitabine treatment yields ~4.5-fold increase in OCT4, ~3-fold increase in mir-145, and ~2-fold increase in mir-302c expression over untreated control. n = 3 for each group.
Figure 4
Figure 4. Doxorubicin treatment increases expression of mir-145 and decreases expression of OCT4 in NTERA-2 cells
NTERA-2 cells, which continually express OCT4, were treated for 16 hours with the indicated doses of doxorubicin, then examined for expression of mir-145 and OCT4 mRNA by qPCR. Expression levels were normalized to untreated NTERA-2 cells, which were arbitrarily set at 1.0. Note, with increasing doses of doxorubicin, mir-145 expression levels increase with a concomitant decrease in OCT4 expression levels. n = 3 for each group.
Figure 5
Figure 5. HSKs treated with decitabine followed by doxorubicin show reduced expression of mir-145 and OCT4
HSKs were treated with doxorubicin alone, decitabine alone, or decitabine for 31 hours followed by doxorubicin for 6 hours. Note, co-treatment reduces the decitabine-induced increase in OCT4 and mir-145 13-fold and 9-fold, respectively. n = 3 for each group.
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