We have determined the nucleotide sequence of the human hsp7O gene and 5' flanking region. The hsp7O gene is transcribed as an uninterrupted primary transcript of 2440 nucleotides composed of a 5' noncoding leader sequence of 212 nucleotides, a 3' noncoding region of 242 nucleotides, and a continuous open reading frame of 1986 nucleotides that encodes a protein with predicted molecular mass of 69,800 daltons. Upstream of the 5' terminus are the canonical TATAAA box, the sequence ATTGG that corresponds in the inverted orientation to the CCAAT motif, and the dyad sequence CTGGAAT/ATTCCCG that shares homology in 12 of 14 positions with the consensus transcription regulatory sequence common to Drosophila heat shock genes. Comparison of the predicted amino acid sequences of human hsp70 with the published sequences of Drosophila hsp70 and Escherichia coli dnaK reveals that human hsp70 is 73% identical to Drosophila hsp70 and 47% identical to E. coli dnaK. Surprisingly, the nucleotide sequences of the human and Drosophila genes are 72% identical and human and E. coli genes are 50% identical, which is more highly conserved than necessary given the degeneracy of the genetic code. The lack of accumulated silent nucleotide substitutions leads us to propose that there may be additional information in the nucleotide sequence of the hsp7O gene or the corresponding mRNA that precludes the maximum divergence allowed in the silent codon positions.The hsp7O gene in eukaryotic cells is a member of a small family of heat shock or stress-induced genes whose expression is regulated at the transcriptional and translational levels by diverse chemical and biological stresses, including inhibitors of energy metabolism, amino acid analogues, heavy metals, anoxia, and heat shock (1, 2). It has been shown recently that the expression of hsp70 is induced by the viral oncogenes of the DNA tumor viruses adenovirus 5, simian virus 40 (SV40), and polyoma and by the mouse rearranged c-myc oncogene (3-5). These oncogenes share at least two interesting features in that their products are subcellularly localized in the nucleus and are implicated in the immortalization of primary cells in culture (6)(7)(8)(9)(10)(11).Although the function of hsp70 is unknown, we can speculate on its role based on the extensive homology between Drosophila hsp70 and the Escherichia coli heat shock protein dnaK. The amino acid sequence of E. coli dnaK derived from its recently determined nucleotide sequence reveals that it is 48% homologous to Drosophila

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 ␤-catenin in normal-appearing intestine. However, ␤-catenin was nuclear in DCAMKL-1-positive cells in adenomas. Thus, nuclear translocation of ␤-catenin distinguishes normal and adenoma stem cells. Targeting DCAMKL-1 may represent a strategy for developing novel chemotherapeutic agents. STEM CELLS 2008;26:630 -637 Disclosure of potential conflicts of interest is found at the end of this article.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43°C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to Human tissue culture cells respond to heat shock, and certain other stimuli, by the induced synthesis of a small set of proteins (molecular weights 100,000, 70,000, and 37,000 [58]). The effect of heat shock on the pattern of protein synthesis in human cells is similar to that for other eucaryotic cells (reviewed in reference 55). This highly conserved response-the activation of a small number of genes and the repression of other normally active genes-has been most intensively studied in Drosophila (1). The altered protein synthetic pattern is, in general, a reflection of both the preferential transcription of heat shock genes and the selected translation of their mRNAs.The major heat shock protein synthesized by eucaryotic cells belongs to a family of 70,000-dalton proteins (HSP70). The conservation of HSP70 among species is revealed by the similar sizes, apparent isoelectric points, and tryptic peptide patterns (64). Indeed, polyclonal antibodies raised against chicken HSP70 cross-react with proteins of similar size from yeast, Drosophila, Xenopus, mice, and humans (32). In Drosophila, the HSP70 multigene family encodes two heat shock proteins, HSP68 and HSP70 (25), and three cognate proteins (27) whose synthesis occurs at normal temperature and which are not heat shock inducible. Drosophila melanogaster has five copies of the HSP70 gene, two at the 87A chromosomal locus and three at the 87C locus (25). Similarly, Saccharomyces cerevisiae contains two copies of the HSP70 gene (28).The sequence conservation of the HSP70 genes among species has been used to isolate the homologous genes from yeast (28) (31). In this study, we describe the structural features of the human HSP70 gene: the organization of the genomic clone and the location of the 5' and 3' termini of the heat shock-induced HSP70 transcript. We examine the expression of the human HSP70 gene in hamster cells and of a chimeric HSP70 bacterial chloramphenicol acetyltransferase (CAT) gene in human cells. MATERIALS AND METHODSGeneral methods. The human genomic lambda library (39) was generously provided by T. Maniatis. Approximately 5 x 105 recombinant phage were screened (22) for sequences homologous to a subclone (plasmid 232.1; 41) containing 1.1 kilobase (kb) of coding sequence adjacent to the 5' end of the Drosophila HSP70 gene. Hybridizing plaques were purified, and DNA was isolated from phage particles banded by equilibrium CsCl centrifugation (42).Genomic DNA was prepared from human placental tissue lysed with sodium dodecyl sulfate (SDS) and digested with proteinase K (8). Subclones of H3-1 were constructed with the v...

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

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 (;-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 ;-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced ;-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 IRinduced signaling required for chromosomal DNA doublestrand break repair, thus resulting in increased cellular radiosensitivity. [Cancer Res 2007;67(7):3010-7]