PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

To proliferate and expand in an environment with limited nutrients, cancer cells co-opt cellular regulatory pathways that facilitate adaptation and thereby maintain tumor growth and survival potential. The endoplasmic reticulum (ER) is uniquely positioned to sense nutrient deprivation stress and subsequently engage signaling pathways that promote adaptive strategies. As such, components of the ER stress-signaling pathway represent potential antineoplastic targets. However, recent investigations into the role of the ER resident protein kinase, RNA-dependent protein kinase (PKR)-like ER kinase (PERK) have paradoxically suggested both pro- and anti-tumorigenic properties. We have used animal models of mammary carcinoma to interrogate the contribution of PERK in the neoplastic process. The ablation of PERK in tumor cells resulted in impaired regeneration of intracellular antioxidants and accumulation of reactive oxygen species triggering oxidative DNA damage. Ultimately, PERK deficiency impeded progression through the cell cycle because of the activation of the DNA damage checkpoint. Our data reveal that PERK-dependent signaling is used during both tumor initiation and expansion to maintain redox homeostasis, thereby facilitating tumor growth.

Brain ischemia and reperfusion activates the eukaryotic initiation factor 2alpha kinase, PERK.

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha). To identify kinases responsible for eIF2alpha phosphorylation [eIF2alpha(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post-ischemic assessment of brain eIF2alpha(P) in mice with homozygous functional knockouts in the genes encoding the heme-regulated eIF2alpha kinase (HRI), or the amino acid-regulated eIF2alpha kinase (GCN2). A 10-fold increase in eIF2alpha(P) was observed in reperfused wild-type mice and in the HRI-/- or GCN2-/- mice. However, in all reperfused groups, the RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eIF2alpha kinase (PERK) exhibited an isoform mobility shift on SDS-PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post-ischemic brain eIF2alpha(P), and in conjunction with our previous report that eIF2alpha(P) is produced in the brain of reperfused PKR-/- mice, provides evidence that PERK is the kinase responsible for eIF2alpha phosphorylation in the early post-ischemic brain.

Complex organization of promoter and enhancer elements regulate the tissue- and developmental stage-specific expression of the Drosophila melanogaster Gld gene.

The Drosophila melanogaster Gld gene has multiple and diverse developmental and physiological functions. We report herein that interactions among proximal promoter elements and a cluster of intronically located enhancers and silencers specify the complex regulation of Gld that underlies its diverse functions. Gld expression in nonreproductive tissues is largely determined by proximal promoter elements with the exception of the embryonic labium where Gld is activated by an enhancer within the first intron. A nuclear protein, GPAL, has been identified that binds the Gpal elements in the proximal promoter region. Regulation of Gld in the reproductive organs is particularly complex, involving interactions among the Gpal proximal promoter elements, a unique TATA box, three distinct enhancer types, and one or more silencer elements. The three somatic reproductive organ enhancers each activate expression in male and female pairs of reproductive organs. One of these pairs, the male ejaculatory duct and female oviduct, are known to be developmentally homologous. We report evidence that the other two pairs of organs are developmentally homologous as well. A comprehensive model to explain the full developmental regulation of Gld and its evolution is presented.

A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha.

A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation. It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity. Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice. We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences. Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain. While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed. Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences. Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate. Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha. Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control.

Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2alpha kinase.

Genomic and cDNA clones homologous to the yeast GCN2 eIF-2alpha kinase (yGCN2) were isolated from Drosophila melanogaster. The identity of the Drosophila GCN2 (dGCN2) gene is supported by the unique combination of sequence encoding a protein kinase catalytic domain and a domain homologous to histidyl-tRNA synthetase and by the ability of dGCN2 to complement a deletion mutant of the yeast GCN2 gene. Complementation of Deltagcn2 in yeast by dGCN2 depends on the presence of the critical regulatory phosphorylation site (serine 51) of eIF-2alpha. dGCN2 is composed of 10 exons encoding a protein of 1589 amino acids. dGCN2 mRNA is expressed throughout Drosophila development and is particularly abundant at the earliest stages of embryogenesis. The dGCN2 gene was cytogenetically and physically mapped to the right arm of the third chromosome at 100C3 in STS Dm2514. The discovery of GCN2 in higher eukaryotes is somewhat unexpected given the marked differences between the amino acid biosynthetic pathways of yeast vs. Drosophila and other higher eukaryotes. Despite these differences, the presence of GCN2 in Drosophila suggests at least partial conservation from yeast to multicellular organisms of the mechanisms responding to amino acid deprivation.

Ultrabithorax and Antennapedia 5' untranslated regions promote developmentally regulated internal translation initiation.

The 5' untranslated regions (UTRs) of the Drosophila Ubx and Antp genes were tested for their ability to promote cap-independent translation initiation. The Ubx and the Antp 5' UTR were inserted between the CAT and lacZ coding sequences in a dicistronic gene and tested for IRES activity in transgenic Drosophila. Northern analysis of the mRNAs showed the presence of the predicted full-length dicistronic mRNAs. High CAT activity was expressed from the first cistron from all of the dicistronic constructs introduced into the fly genome. The dicistronic transgenic strains bearing the Ubx and Antp IRES elements expressed significant levels of beta-galactosidase (betaGAL) from the second cistron whereas little or no betaGAL was expressed in the controls lacking the IRESs. In situ analysis of betaGAL expression in the transgenic strains indicates that expression of the second cistron is spatially and temporally regulated. Although the developmental patterns of expression directed by the Antp and Ubx IRESs overlap, they exhibit several differences indicating that these IRESs are not functionally equivalent.

Mutations at the Ser50 residue of translation factor eIF-2alpha dominantly affect developmental rate, body weight, and viability of Drosophila melanogaster.

Phosphorylation of the translation initiation factor eIF-2alpha downregulates protein synthesis by sequestering the guanylate exchange factor eIF-2B. The importance of this regulation has been demonstrated in the context of stress and virally induced repression of protein synthesis but has not been investigated relative to the control of protein synthesis during development. Transgenic Drosophila strains bearing aspartic acid or alanine substitutions at the presumed regulatory phosphorylation site (Ser50) of Drosophila eIF-2alpha were established. The expression of the eIF-2alpha mutant transgenes, under the transcriptional control of the hsp70 promoter, was induced at various times during development to assess the developmental and biochemical effects. Flies bearing the aspartic acid eIF-2alpha mutant (HD) transgene displayed a slow growth phenotype and small body size. Repeated induction of the HD transgene resulted in cessation of development. In contrast, flies bearing the alanine eIF-2alpha mutant (HA) displayed a fast growth phenotype and females were significantly larger than nontransgenic control sisters. The HD transgenic flies exhibit a relatively lower level of global protein synthesis than the HA transgenic flies, although the difference is statistically insignificant.

A somatic reproductive organ enhancer complex activates expression in both the developing and the mature Drosophila reproductive tract.

The Glucose dehydrogenase (Gld) gene is highly expressed in the mature Drosophila reproductive tract. Unlike several other Drosophila genes which function in reproductive physiological processes, Gld is also expressed extensively in the developing reproductive tract during metamorphosis. Proximal promoter elements drive Gld expression in a variety of tissues throughout development, but not in the reproductive tract. Herein, we have identified a somatic reproductive organ enhancer complex (SREC) containing multiple redundant enhancer modules in Gld intron I (+639 to +3906 nt). The SREC, in combination with the Gld promoter, activates beta galactosidase reporter gene expression in both the developing and the mature reproductive tract. The SREC activates a heterologous hsp70 promoter in the ejaculatory duct, but not in other reproductive tract tissues, suggesting that the SREC acts synergistically with Gld promoter proximal elements. Through deletion analysis we have delimited a 361-nt region of the SREC that is involved in ejaculatory duct/oviduct-specific expression. The ejaculatory duct/oviduct enhancer retains the ability to activate expression in both the developing and the mature reproductive tract, suggesting that the same basic enhancer elements activate Gld expression during metamorphosis and in adults. A model of the evolution of Gld expression in the ejaculatory duct and oviduct is presented.

Heat shock effects on phosphorylation of protein synthesis initiation factor proteins eIF-4E and eIF-2 alpha in Drosophila.

Heat shock of mammalian cells causes changes in initiation factor phosphorylation that likely contribute to or cause the translation reprogramming characteristic of heat shock. In these investigations we have carried out a parallel analysis of Drosophila, focusing on eIF-4E and eIF-2 alpha. eIF-4E plus associated proteins was purified from lysates by m7GTP-Sepharose chromatography. A minor fraction (< 10%) of eIF-4E is phosphorylated under normal growth conditions, and phosphorylation decreases during heat shock. Drosophila eIF-2 alpha has been identified by in vitro translation of T7 RNA polymerase-transcribed mRNA, and immunoblotting with anti-Drosophila eIF-2 alpha antiserum. 32P-labeling analysis (unfractionated cell lysates and immunoprecipitates) detects phosphorylated eIF-2 alpha, whose amount increases approximately 2-3-fold upon heat shock. Immunoblotting analysis of two-dimensional gel-resolved proteins to determine the mass fraction of eIF-2 alpha phosphorylated detects a single eIF-2 alpha spot in both normal temperature and heat shocked cells, indicating less than 5% phosphorylation after and before heat shock. Staining quantification is consistent with this low prevalence. A major phosphoprotein which copurifies with eIF-4E on m7GTP-Sepharose shows decreased overall phosphorylation and decreased association with eIF-4E following heat shock. Several distinctive characteristics of this phosphoprotein suggest it is Drosophila eIF-4B.

Isolation and characterization of the Drosophila melanogaster gene encoding translation-initiation factor eIF-2 beta.

Drosophila melanogaster cDNA clones encoding the beta subunit of translation initiation factor 2 (eIF-2) were isolated and sequenced. The longest cDNA predicts a protein of 312 amino acids (aa), which possesses a putative RNA-binding motif and a highly charged N-terminal region composed of three basic polylysine blocks. The aa sequence comparison of D. melanogaster eIF-2 beta with its human and yeast counterparts demonstrates a high degree of similarity, especially within the C-terminal region. Northern analysis indicates quasi-constitutive expression of eIF-2 beta throughout D. melanogaster development.

An evolutionarily conserved palindrome in the Drosophila Gld promoter directs tissue-specific expression.

A conserved palindromic sequence (Gpal) in the promoter region of the Drosophila Gld directs expression of a heterologous reporter gene in transgenic flies to the anterior spiracular glands of third instar larvae and to the ejaculatory bulb of adult males. The Gld gene is normally expressed at high levels in the anterior spiracular glands but is not expressed in the ejaculatory bulb of Drosophila melanogaster. However, Gld promoters from other Drosophila species contain the Gpal element and express glucose dehydrogenase (GLD) in the adult male ejaculatory bulb. A gene fusion composed of the D. melanogaster Gld promoter and the lacZ gene is expressed in the anterior spiracular glands of transgenic larvae. Mutations of the Gpal sequence element in this gene fusion block expression of beta-galactosidase in the anterior spiracular gland. Together these experiments demonstrate that Gpal is necessary and sufficient for tissue-specific expression in the anterior spiracular glands. Based upon the tissue distribution and function of GLD, it is speculated that expression of GLD in the anterior spiracular glands represents the ancestral state and that GLD expression in other tissues arose as a fortuitous consequence of a shared combinatorial regulatory network.

Isolation and characterization of the Drosophila melanogaster eIF-2 alpha gene encoding the alpha subunit of translation initiation factor eIF-2.

Genomic and cDNA clones encoding the Drosophila melanogaster alpha-subunit of translational initiation factor 2 (eIF-2 alpha) were isolated. The D. melanogaster eIF-2 alpha gene encodes a 341 amino-acid (aa) protein that shares 57 and 44% identity to its human and yeast homologues, respectively. The regulatory phosphorylation site at Ser50 is embedded in a segment of 19 conserved aa residues. Analysis of the genomic DNA and cDNA clones indicated that eIF-2 alpha is a single-copy gene and its coding region is interrupted by a 260-bp intron. The D. melanogaster eIF-2 alpha mRNA is 1350 nt in length and is expressed throughout development.

Correlated evolution of the cis-acting regulatory elements and developmental expression of the Drosophila Gld gene in seven species from the subgroup melanogaster.

The tissue-specific expression patterns of glucose dehydrogenase (GLD) exhibit a high degree of interspecific variation in the adult reproductive tract among the species in the genus Drosophila. We chose to focus on the evolution of GLD expression and the evolution of the Gld promoter in seven closely related species in the melanogaster subgroup as a means of elucidating the relationship of changes in cis-acting regulatory elements in the Gld promoter region with changes in tissue-specific expression. Although little variation in tissue-specific patterns of GLD was found in nonreproductive tissues during development, a surprisingly high level of variation was observed in the expression of GLD in both developing and mature reproductive organs. In some cases this variation is correlated with changes in sequence elements in the Gld promoter which were previously shown to direct tissue-specific expression in the reproductive tract. In particular D. teissieri adult males do not express GLD in their ejaculatory ducts, atypical of the melanogaster subgroup species. The Gld promoter region of D. teissieri specifically lacks all three of the TTAGA regulatory elements present in D. melanogaster. The TTAGA elements were previously shown to direct reporter gene expression to the ejaculatory duct. Together these data suggest the absence or presence of the TTAGA elements may be responsible for variation in the absence or presence of GLD in the ejaculatory duct among species.

Tissue-specific regulatory elements of the Drosophila Gld gene.

Putative cis-acting regulatory elements immediately upstream of the Gld promoter were identified by comparative analysis of three Drosophila species. A 509 bp region containing these elements and the Gld promoter region was shown to confer tissue-specific regulation to a reporter gene similar to the pattern observed for Gld mRNA and protein. A dispersed repeat with a core motif of TTAGA was also capable of directing the expression of a reporter gene to several epidermally derived tissues in which GLD is normally expressed. These tissues include male and female somatic reproductive organs. The TTAGA elements and a palindromic element act antagonistically to block expression of reporter gene in some tissues. Previously reported mutations of the heat shock response element resulted in the creation of three TTAGA elements. This mutated hsp70 promoter directs expression of a reporter gene to many of the same tissues as does the Gld TTAGA elements. We have found TTAGA elements near the promoter of two other genes which show an identical expression pattern in the male ejaculatory duct as Gld and the mutant hsp70.

Evolution of the expression of the Gld gene in the reproductive tract of Drosophila.

During the preadult development of Drosophila melanogaster, the GLD (glucose dehydrogenase) gene (Gld) is expressed in a variety of tissues, including the immature reproductive tract. At the adult stage the expression of Gld becomes largely restricted to the reproductive tract of males and females. We examined the expression of GLD in the adult reproductive tract of 50 species in the genus Drosophila, as well as in those of a few representative species from four other closely related genera. GLD exhibits considerable organ-specific diversity in the reproductive tract of males and females. Among these species, five male GLD phenotypes and six female GLD phenotypes were found. In contrast, the preadult expression of GLD in representative species from each distinct adult pattern type was determined and found to be highly conserved in both the immature reproductive tract and non-reproductive organs. Moreover, the set of reproductive organs that express GLD during preadult development is equivalent to the sum of the five male and six female adult GLD phenotypes. To initially define the contribution of cis- versus trans-acting factors responsible for differences in adult GLD expression between two of these species--D. melanogaster and D. pseudoobscura--we transferred the D. pseudoobscura Gld to the genome of D. melanogaster and investigated its expression. GLD expression patterns of these transformants displayed characteristics that are unique to both species, suggesting the presence of both cis- and trans-acting differences between these two species.

The Drosophila melanogaster stranded at second (sas) gene encodes a putative epidermal cell surface receptor required for larval development.

Several lethal mutations were identified previously in the 84BD interval of the Drosophila melanogaster third chromosome (Lewis et al., 1980; Cavener et al., 1986b). We have examined the l(3)84Cd complementation group and found that mutants exhibit novel cuticular defects and die during larval development. The lethal phase occurs during the first larval molt or subsequently during the second instar larval stage; hence, we have named the gene stranded at second (sas). There are no apparent effects on the rate of development of embryos or first instar larvae. Second instar larvae which survive the molt exhibit a marked reduction in growth and eventually die as small second instar larvae. Incomplete penetrance in some weak sas alleles can yield fertile adults. In addition to the lethal phenotype, a segmentally repeated pattern of tanned spots is found within the ventral setal belts of mutant larvae. The position of the spots is always either between the fourth and fifth row of setae (cuticular projections) or between the first and second row of setae. The spots are adjacent to the muscle attachment sites in the setal belt region. Another common larval phenotype is the abnormal tanning of the ventral surface of the pharynx. The sas gene was cloned, and both the cuticular tanning and the larval lethal phenotypes were complemented by P-element-mediated transformation with a genomic DNA-cDNA construct. Three major sas transcripts are expressed throughout development in cuticle secreting epidermal tissues. The sas transcripts show stage- and tissue-specific patterns of expression with switches in transcript patterns occurring at the molts. The inferred 1348-amino-acid sequence suggests that sas encodes a cell surface protein which functions as a receptor. The putative extracellular region contains four tandem repeats of a cysteine-rich motif which is similar to a cysteine pattern present in procollagen and in thrombospondin. Following this region are at least three copies of a fibronectin type III class repeat. The short (35 amino acids) intracellular domain contains a sequence (NPXY) that has been implicated in endocytosis via coated pits.

Transgenic animal studies on the evolution of genetic regulatory circuitries.

The ability to transfer genes from one species to another provides a powerful method to study genetic regulatory differences between species in a homogeneous genetic background. A survey of several transgenic animal experiments indicates that the vast majority of regulatory differences observed between species are due to differences in the cis-acting elements associated with the genes under study. A corollary is that in almost all cases the host species provides the necessary regulatory proteins for expression of the transgenes in specific tissues in which the endogenous homolog is not expressed. Although the details of the cis-acting differences are unknown for most cases, it appears that these differences may consist of the acquisition or loss of unique elements or subtle variation of conserved elements. It is unknown whether much of this variation is directly related to adaptive evolution. The identification of the promoter/enhancer elements responsible for these differences is an important first step in examining the functional significance of this variation.

GMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities.

Sequence comparison of Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase and Hansenula polymorpha methanol oxidase indicates that these four diverse flavoproteins are homologous, defining a new family of proteins named the GMC oxidoreductases. These enzymes contain a canonical ADP-binding beta alpha beta-fold close to their amino termini as found in other flavoenzymes. This domain is encoded by a single exon of the D. melanogaster glucose dehydrogenase gene.

Analysis of the PvuII restriction fragment-length polymorphism and exon structure of the estrogen receptor gene in breast cancer and peripheral blood.

The presence of estrogen receptor (ER) is a well-known predictor of clinical outcome in human breast cancer. We examined the ER gene in 26 primary breast cancers (14 ER-positive, 12 ER-negative) to determine if alterations of the gene are associated with the ER-negative status. In tumor biopsies and peripheral blood DNA obtained from the same patients we analyzed the ER exon structure using polymerase chain reaction amplification, restriction endonuclease digestion, and agarose gel electrophoresis. All blood and tumor samples, regardless of ER status, showed a complete set of eight exons of normal sizes, ruling out deletions or rearrangements of the ER gene in excess of +/- 20 nucleotides. Previous reports indicate that the two-allele ER PvuII polymorphism could be associated with ER expression in breast cancer (Hill et al., Cancer Res., 49: 145-148, 1989) as well as with patient age at time of tumor diagnosis (Parl et al., Breast Cancer Res. Treat., 14: 57-64, 1989). We localized the PvuII polymorphism in intron 1, 0.4 kilobase upstream of exon 2. Sequence analysis showed the polymorphism to result from a point mutation (T----C) at the fifth position of the restriction site (CATCTG). We determined the PvuII restriction fragment-length polymorphism genotype in 257 primary breast cancers and 140 peripheral blood DNA samples obtained from women without breast cancer. The results indicate that the PvuII polymorphism is not associated with ER content or patient age at tumor diagnosis.