Culture of endodermal stem/progenitor cells of the mouse tongue

The tongue represents a very accessible source of tissue-specific epithelial stem cells of endodermal origin. However, little is known about the properties of these cells and the mechanisms regulating their proliferation and differentiation. Foxa2, an endodermal marker, is expressed throughout the tongue epithelium during embryonic development but becomes confined to a minority of basal cells and some taste bud sensory cells in the adult tongue. Using a previously described line of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of a human keratin 5 promoter region (Krt5-eGFP), we have isolated a subpopulation of cells in the basal epithelial layer of the mouse tongue with a high efficiency of generating holoclones of undifferentiated cells in culture with a feeder layer. Krt5-GFP(hi) cells can both self renew and give rise to differentiated stratified keratinized epithelial cells when cultured on an air-liquid interface.     

NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation

Phosphorylation of replaceable histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called gamma-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP) which could potentially increase the intracellular level of NAD+ and on the level of gamma-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NAD+ in different doses immediately after IR causes an increased level of gamma-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that it could be a relationship between intracellular NAD+ content and DNA damage response in vivo.     

Exogenous NADP increases the level of histone H2AX phosphorylation in mouse heart cells after ionizing radiation

Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γ-H2AX can be used as an effective marker for DSBs repair and DNA damage response. Using Western blotting and immunohistochemistry techniques we have studied here the influence of exogenous nicotinamide adenine dinucleotide phosphate (NADP), which can potentially increase the level of intracellular NAD⁺, on the level of γ-H2AX formation in mouse heart cells after ionizing radiation (IR). We have found that injection of NADP in different doses immediately after IR causes an increased level of γ-H2AX in mouse heart cells 20 min after IR at the dose of 3 Gy compared to control mice after IR exposure. It indicates that there could be a relationship between intracellular NAD⁺ content and DNA damage response in vivo.     

Dexrazoxane ameliorates doxorubicin-induced injury in mouse ovarian cells

Doxorubicin (DXR) is a frontline chemotherapy agent implicated in unintended ovarian failure in female cancer survivors. The fertility preservation techniques currently available for cancer patients are often time and cost prohibitive and do not necessarily preserve endocrine function. There are no drug-based ovary protection therapies clinically available. This study provides the first investigation using dexrazoxane (Dexra) to limit DXR insult in ovarian tissue. In KK-15 granulosa cells, a 3-h DXR treatment increased double-strand (ds) DNA breaks 40%-50%, as quantified by the neutral comet assay, and dose-dependent cytotoxicity. Dexra exhibited low toxicity in KK-15 cells, inducing no DNA damage and less than 20% cell loss. Cotreating KK-15 cells with Dexra prevented acute DXR-induced dsDNA damage. Similarly, Dexra attenuated the DXR-induced 40%-65% increase in dsDNA breaks in primary murine granulosa cells and cells from in vitro cultured murine ovaries. DXR can cause DNA damage either through a topoisomerase II-mediated pathway, based on DXR intercalation into DNA, or through oxidative stress. Cotreating KK-15 cells with 2 μM Dexra was sufficient to prevent DXR-induced, but not H(2)O(2)-induced, DNA damage. These data indicated the protective effects are likely due to Dexra's inhibition of topoisomerase II catalytic activity. This putative protective agent attenuated downstream cellular responses to DXR, preventing H2AFX activation in KK-15 cells and increasing viability as demonstrated by increasing the DXR lethal dose in KK-15 cells 5- to 8-fold (LD(20)) and primary murine granulosa cells 1.5- to 2-fold (LD(50)). These data demonstrate Dexra protects ovarian cells from DXR insult and suggest that it is a promising tool to limit DXR ovarian toxicity in vivo.     

MicroRNA-30 inhibits antiapoptotic factor Mcl-1 in mouse and human hematopoietic cells after radiation exposure

We previously reported that microRNA-30 (miR-30) expression was initiated by radiation-induced proinflammatory factor IL-1β and NFkB activation in mouse and human hematopoietic cells. However, the downstream effectors of miR-30 and its specific role in radiation-induced cell death are not well understood. In the present study, we evaluated effects of radiation on miR-30 expression and activation of intrinsic apoptotic pathway Bcl-2 family factors in in vivo mouse and in vitro human hematopoietic cells. CD2F1 mice and human CD34+ cells were exposed to different doses of gamma-radiation. In addition to survival studies, mouse blood, bone marrow (BM) and spleen cells and human CD34+ cells were collected at 4 h, and 1, 3 and 4 days after irradiation to determine apoptotic and stress response signals. Our results showed that mouse serum miR-30, DNA damage marker γ-H2AX in BM, and Bim, Bax and Bak expression, cytochrome c release, and caspase-3 and -7 activation in BM and/or spleen cells were upregulated in a radiation dose-dependent manner. Antiapoptotic factor Mcl-1 was significantly downregulated, whereas Bcl-2 was less changed or unaltered in the irradiated mouse cells and human CD34+ cells. Furthermore, a putative miR-30 binding site was found in the 3′ UTR of Mcl-1 mRNA. miR-30 directly inhibits the expression of Mcl-1 through binding to its target sequence, which was demonstrated by a luciferase reporter assay, and the finding that Mcl-1 was uninhibited by irradiation in miR-30 knockdown CD34+ cells. Bcl-2 expression was not affected by miR-30. Our data suggest miR-30 plays a key role in radiation-induced apoptosis through directly targeting Mcl-1in hematopoietic cells.     

Potentiation of thermal injury in mouse cells by interferon

Mouse cells, when exposed to high temperature (43 degrees), shut off overall protein synthesis and continue to synthesize "heat shock proteins". Such heat shocked cells, upon reincubation at 37 degrees C, recover and proliferate. However, when mouse cells are pretreated with mouse interferon (IFN) and then exposed to 43 degrees, more than 99% of the cell population fail to recover. Synthesis of the major heat shock protein is unaffected in cells treated with IFN. Experiments designed to assess the role of intracellular glutathione (GSH) during cells' recovery from hyperthermia indicated that there is an irreversible depletion of glutathione when IFN treated cells are heat shocked. Neither depletion of GSH, nor potentiation of thermal injury was observed in a IFN-resistant line of mouse cells.     

Radiation damage to and recovery of mouse T-cells. Dynamics of suppressor cells after the effects of radiation

Suppressor and helper T-cells precursors, similar in radiosensitivity (D0 = 2.16 and 2.25, respectively), are restored almost synchronously reaching a normal level by the end of the 2nd month after irradiation. Suppressor macrophage precursors remain intact at doses of up to 7 Gy: one month after irradiation their level decreases perhaps because of the death of the radiosensitive precursors. The suppressors damage is one of the factors decelerating the reverse development of the immune response after irradiation.     

The assessment of recovery of the intestine after acute radiation injury

Several aspects of intestinal function and morphology are affected by acute radiation damage, including changes in the activity of proliferative cells in the crypts, immune cell populations, and the transport of various substrates. This study was designed to compare the time course of the recovery of intestinal proliferation, transport, and leukocyte population following radiation injury. Rats received a single dose of 6 Gy to the abdomen from a 137Cs source and were studied 3, 7, and 14 days later. No changes in the passive uptake of L-glucose or D-leucine were observed in the jejunum. Active transport of D-glucose and maximal water uptake were reduced at 3 days but had returned to normal by 7 days, whereas L-leucine uptake required more than 7 days to return to control levels. Mucosal permeability, assessed by an in vivo potential difference technique, remained increased 7 days after irradiation. Ornithine decarboxylase, an indicator of DNA synthetic activity, was elevated following radiation treatment and remained so even after 14 days. By comparison, myeloperoxidase activity, used as a quantitative monitor of granulocyte numbers, was still reduced after 7 days. These data indicate that while certain parameters of gut function may return to normal soon after radiation injury, the recovery of other factors is more prolonged. Thus the return of transport function to normal values post irradiation may be viewed as an adaptive change rather than simply the recovery of the tissue.