Clayton R. Hunt

Identification of a Novel Putative Gastrointestinal Stem Cell and Adenoma Stem Cell Marker, Doublecortin and CaM Kinase‐Like‐1, Following Radiation Injury and in Adenomatous Polyposis Coli/Multiple Intestinal Neoplasia Mice

In the gut, tumorigenesis arises from intestinal or colonic crypt stem cells. Currently, no definitive markers exist that reliably identify gut stem cells. Here, we used the putative stem cell marker doublecortin and CaM kinase‐like‐1 (DCAMKL‐1) to examine radiation‐induced stem cell apoptosis and adenomatous polyposis coli (APC)/multiple intestinal neoplasia (min) mice to determine the effects of APC mutation on DCAMKL‐1 expression. Immunoreactive DCAMKL‐1 staining was demonstrated in the intestinal stem cell zone. Furthermore, we observed apoptosis of the cells negative for DCAMKL‐1 at 6 hours. We found DNA damage in all the cells in the crypt region, including the DCAMKL‐1‐positive cells. We also observed stem cell apoptosis and mitotic DCAMKL‐1‐expressing cells 24 hours after irradiation. Moreover, in APC/min mice, DCAMKL‐1‐expressing cells were negative for proliferating cell nuclear antigen and nuclear β‐catenin in normal‐appearing intestine. However, β‐catenin was nuclear in DCAMKL‐1‐positive cells in adenomas. Thus, nuclear translocation of β‐catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL‐1 may represent a strategy for developing novel chemotherapeutic agents.

Genomic Instability and Enhanced Radiosensitivity in Hsp70.1-and Hsp70.3 -Deficient Mice

ABSTRACT Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3−/− mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3−/− mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3−/− mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3−/− mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3+/+ cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3−/− and Hsp70.1/3+/+ fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3−/− cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3−/− cells in comparison to Hsp70.1/3+/+ cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.

Hyperthermia Activates a Subset of Ataxia-Telangiectasia Mutated Effectors Independent of DNA Strand Breaks and Heat Shock Protein 70 Status

All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.

The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation.

Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase IIalpha and IIbeta genes. In HeLa cells synchronized by mitotic shake-off, topoisomeraseII alpha mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G1 phase. Also detected was a corresponding increase in topoisomerase IIalpha protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G2 phases. Topoisomerase IIalpha expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase IIalpha expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, the half-life of topoisomerase IIalpha mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase IIalpha mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45 degrees C for 15 min) caused the accumulated topoisomerase IIalpha mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase IIalpha expression and cell cycle progression delays in late S/G2.

Proto-Oncogene mRNA Levels and Activities of Multiple Transcription Factors in C3H 10T 1/2 Murine Embryonic Fibroblasts Exposed to 835.62 and 847.74 MHz Cellular Phone Communication Frequency Radiation

This study was designed to determine whether two differently modulated radiofrequencies of the type generally used in cellular phone communications could elicit a general stress response in a biological system. The two modulations and frequencies studied were a frequency-modulated continuous wave (FMCW) with a carrier frequency of 835.62 MHz and a code division multiple-access (CDMA) modulation centered on 847.74 MHz. Changes in proto-oncogene expression, determined by measuring Fos, Jun, and Myc mRNA levels as well as by the DNA-binding activity of the AP1, AP2 and NF-kappaB transcription factors, were used as indicators of a general stress response. The effect of radiofrequency exposure on proto-oncogene expression was assessed (1) in exponentially growing C3H 10T 1/2 mouse embryo fibroblasts during their transition to plateau phase and (2) during transition of serum-deprived cells to the proliferation cycle after serum stimulation. Exposure of serum-deprived cells to 835.62 MHz FMCW or 847.74 MHz CDMA microwaves (at an average specific absorption rate, SAR, of 0.6 W/kg) did not significantly change the kinetics of proto-oncogene expression after serum stimulation. Similarly, these exposures did not affect either the Jun and Myc mRNA levels or the DNA-binding activity of AP1, AP2 and NF-kappaB in exponential cells during transit to plateau-phase growth. Therefore, these results suggest that the radiofrequency exposure is unlikely to elicit a general stress response in cells of this cell line under these conditions. However, statistically significant increases (approximately 2-fold, P = 0.001) in Fos mRNA levels were detected in exponential cells in transit to the plateau phase and in plateau-phase cells exposed to 835.62 MHz FMCW microwaves. For 847.74 MHz CDMA exposure, the increase was 1.4-fold (P = 0.04). This increase in Fos expression suggests that expression of specific genes could be affected by radiofrequency exposure.

Cell cycle checkpoint defects contribute to genomic instability in PTEN deficient cells independent of DNA DSB repair

Chromosomes in PTEN deficient cells display both numerical as well as structural alterations including regional amplification. We found that PTEN deficient cells displayed a normal DNA damage response (DDR) as evidenced by the ionizing radiation (IR)-induced phosphorylation of Ataxia Telangiectasia Mutated (ATM) as well as its effectors. PTEN deficient cells also had no defect in Rad51 expression or DNA damage repair kinetics post irradiation. In contrast, caffeine treatment specifically increased IR-induced chromosome aberrations and mitotic index only in cells with PTEN, and not in cells deficient for PTEN, suggesting that their checkpoints were defective. Furthermore, PTEN-deficient cells were unable to maintain active spindle checkpoint after taxol treatment. Genomic instability in PTEN deficient cells could not be attributed to lack of PTEN at centromeres, since no interaction was detected between centromeric DNA and PTEN in wild type cells. These results indicate that PTEN deficiency alters multiple cell cycle checkpoints possibly leaving less time for DNA damage repair and/or chromosome segregation as evidenced by the increased structural as well as numerical alterations seen in PTEN deficient cells.

Redox Factor-1 (Ref-1) Mediates the Activation of AP-1 in HeLa and NIH 3T3 Cells in Response to Heat Shock

The early response genes, c-Fos and c-Jun, are induced by environmental stress and are thought to modulate injury processes via the induction of AP-1-dependent target genes. AP-1 activation is thought to be regulated by changes in intracellular oxidation/reduction reactions involving the redox factor-1 (Ref-1) protein. In this study, NIH 3T3 and HeLa cells were used to determine whether heat shock induces the AP-1 transcription factor via signaling pathways involving Ref-1. Reverse transcriptase-polymerase chain reaction analysis and immunoblotting demonstrated that c-Fos and c-Jun were induced 2–10 h following heat shock, and this induction was accompanied by an increase in AP-1 DNA binding. Electrophoretic mobility shift assay extracts immunodepleted of Ref-1 protein demonstrated that the increase in AP-1 DNA-binding activity following heating was dependent upon the presence of Ref-1 and that Ref-1 regulates inducible, but not basal, AP-1 DNA-binding activity. This was confirmed by the restoration of heat-inducible DNA binding upon addition of Ref-1 to immunodepleted extracts. The ability of Ref-1 from heated cells to stimulate AP-1 DNA binding was abolished by chemical oxidation and restored by chemical reduction. These results indicate that heat shock activates c-Fos/c-Jun gene expression and AP-1 DNA binding and suggests that redox-sensitive signal transduction pathways involving Ref-1 may mediate heat-induced alterations in AP-1 activation.

Histone Modifications and DNA Double-Strand Break Repair after Exposure to Ionizing Radiations

Ionizing radiation exposure induces highly lethal DNA double-strand breaks (DSBs) in all phases of the cell cycle. After DSBs are detected by the cellular machinery, these breaks are repaired by either of two mechanisms: (1) nonhomologous end joining (NHEJ), which re-ligates the broken ends of the DNA and (2) homologous recombination (HR), that makes use of an undamaged identical DNA sequence as a template to maintain the fidelity of DNA repair. DNA DSB repair must occur within the context of the natural cellular DNA structure. Among the major factors influencing DNA organization are specific histone and nonhistone proteins that form chromatin. The overall chromatin structure regulates DNA damage responses since chromatin status can impede DNA damage site access by repair proteins. During the process of DNA DSB repair, several chromatin alterations are required to sense damage and facilitate accessibility of the repair machinery. The DNA DSB response is also facilitated by hierarchical signaling networks that orchestrate chromatin structural changes that may coordinate cell-cycle checkpoints involving multiple enzymatic activities to repair broken DNA ends. During DNA damage sensing and repair, histones undergo posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation and ubiquitylation. Such histone modifications represent a histone code that directs the recruitment of proteins involved in DNA damage sensing and repair processes. In this review, we summarize histone modifications that occur during DNA DSB repair processes.

Induction of heat shock protein 70 inhibits ischemic renal injury

Heat shock protein 70 (Hsp70) is a potent antiapoptotic agent. Here, we tested whether it directly regulates renal cell survival and organ function in a model of transient renal ischemia using Hsp70 knockout, heterozygous, and wild-type mice. The kidney cortical Hsp70 content inversely correlated with tubular injury, apoptosis, and organ dysfunction after injury. In knockout mice, ischemia caused changes in the activity of Akt and glycogen synthase kinase 3-β (kinases that regulate the proapoptotic protein Bax), increased active Bax, and activated the proapoptotic protease caspase 3. As these changes were significantly reduced in the wild-type mice, we tested whether Hsp70 influences ischemia-induced apoptosis. An Hsp70 inducer, geranylgeranylacetone, increased Hsp70 expression in heterozygous and wild-type mice, and reduced both ischemic tubular injury and organ dysfunction. When administered after ischemia, this inducer also decreased tubular injury and organ failure in wild-type mice but did not protect the knockout mice. ATP depletion in vitro caused greater mitochondrial Bax accumulation and death in primary proximal tubule cells harvested from knockout compared with wild-type mice and altered serine phosphorylation of a Bax peptide at the Akt-specific target site. In contrast, lentiviral-mediated Hsp70 repletion decreased mitochondrial Bax accumulation and rescued Hsp70 knockout cells from death. Thus, increasing Hsp70 either before or after ischemic injury preserves renal function by attenuating acute kidney injury.

MOF Phosphorylation by ATM Regulates 53BP1-Mediated Double-Strand Break Repair Pathway Choice

Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase.