Viral manipulation of cell death.

Elucidation of the mechanisms behind cell death has brought with it an appreciation for viral strategies that target these pathways as a means to promote viral propagation while avoiding or slowing the host immune response. Several redundant anti-viral pathways have evolved in eukaryotic cells that are designed to minimize the damage due to viral infection while quickly clearing the invading pathogen. Cell death is a commonly employed immune defense against viral infection, and many viruses potently induce or suppress cell death during infection. The past decade has seen an incredible increase in our understanding of how cell death assists in host immune response, as well as how viruses have evolved to hijack or disengage these systems. By targeting components of host cell death pathways, viruses have developed the ability to control host survival and death, ensuring efficient propagation while inactivating or avoiding the immune system consequences of infection. This review focuses on the most recent and important advances in our understanding of how a wide range of viruses manipulate the survival and death of their hosts.

Clusterin protein diversity in the primate eye.

PURPOSE: The clusterin gene encodes a multi-functional protein that has been identified in different tissues, including a number of different eye tissues, primarily in the mouse and to a much lesser extent in humans. Clusterin has been implicated in a number of cellular processes such as lipid transport, membrane integrity, apoptosis, and neurodegeneration, all of which could be important to the biology of the eye. In the current communication, we provide data that confirms the expression of clusterin in a number of different human eye tissues and establishes the expression profile of this gene in monkey derived eye tissues. The issue that we sought to examine is whether a broad profile of clusterin expression in the eye is consistent in primates (monkey and human). METHODS: The majority of our study was done using monkey eye tissues. Where possible, we have used human tissues in order to confirm published findings. Northern and western analysis was performed using tissues derived from monkey eyes. In situ hybridization and immunochemistry were carried out on human eye sections. RESULTS: Clusterin mRNA is expressed in primate lens, cornea, limbus, sclera, orbital muscle, ciliary body, retina, RPE/choroid, and RPE cells in culture. Western analysis revealed that two major groups of clusterin exist in the eye, a high molecular weight group (>100 kDa) and a second group consisting of at least five clusterin species that are all approximately 80 kDa. Analysis of conditioned media from RPE cells cultured on permeable supports suggests that different forms of clusterin display alternative patterns of secretion. CONCLUSIONS: Clusterin is expressed in a broad range of eye tissues in both human and monkey, suggesting that this is a characteristic feature in primates. We demonstrate for the first time that a diverse number of clusterin isoforms were observed in monkey eye tissues by western analysis. Meanwhile, the molecular size of clusterin mRNA detected in the array of tissues are identical in size, suggesting that the nature of the diversity in clusterin forms is due to post-translational modifications. In addition, new insights were made in defining clusterin expression in ciliary body, cornea, and the retinal pigment epithelium.

Rapid early onset lymphocyte cell death in mice resistant, but not susceptible to Leishmania major infection.

Leishmania major (Lm) infection in mice is a prototypical model for the role of immune deviation in disease resistance. Resistant strains of mice develop a Th1 response to Lm infection, distinguished by secretion of IL-12 and interferon gamma. In contrast, susceptible strains display sustained IL-4 expression characteristic of a Th2 response. However, when mechanisms of cell death are blocked, mice display a susceptible phenotype even in the presence of a strong Th1 response, suggesting that cell death, and not cytokine bias, may be an important factor in disease resistance. Here, we investigated this hypothesis by comparing lymphocyte cellularity, cell death and Fas expression in resistant CBA and susceptible BALB/c mice during the course of Lm infection. We found that delayed onset of cell death and late Fas induction correlated with massive lymphocyte accumulation and susceptibility to leishmaniasis, while early cell death and rapid Fas induction occurred in resistant mice.

Cyclin-dependent kinase 5 is associated with apoptotic cell death during development and tissue remodeling.

In a series of studies to more precisely localize the cellular sites of expression of the cyclin-dependent kinase (Cdk) family members in reproductive organs, we observed a striking expression of Cdk5 in atretic follicles in the ovary, particularly in granulosa cells that appeared to be dying. We determined that these granulosa cells were undergoing apoptotic cell death using the in situ DNA fragmentation assay. To extend the generality of the association of Cdk5 with apoptotic cells, we examined its expression as it correlated with the detection of apoptosis in a number of developmental paradigms, including regions of the embryonic nervous system, the developing eye, and the developing limb. Finally, the association of apoptosis and Cdk5 expression and associated kinase activity was examined in the limb and in an induced cell death system, that of androgen withdrawal-induced regression of the prostate gland in male mice. These observations provide new insight into the possible function of this novel Cdk during both differentiation and apoptotic cell death.

Cell death/apoptosis: normal, chemically induced, and teratogenic effect.

Cell death is an integral part of a variety of biological processes including cell proliferation, differentiation, and morphogenesis. We review here the morphological and biochemical nature as well as the genetic basis for cell death during normal and abnormal development. Most often referred to in normal development as programmed cell death, this controlled process determines the size, patterning, and function of many tissues. The importance of its proper genetic regulation is demonstrated by the discovery of cell death-specific genes and the several disorders including cancer and teratogenesis that result from repression or enhancement of cell death. In our studies we employed the developing mouse limb, which provides a defined window of active cell death, to elucidate mechanisms of cell death. We have developed markers that reveal in the developing normal limb an apoptotic morphology with phagocytosis and DNA fragmentation. In the limb deformity mutant Hammertoe there is a defective (restricted) cell death pattern, but the morphology remains apoptotic. By the use of these markers, we were able to observe that the teratogen retinoic acid produced enhanced apoptotic cell death. Most interestingly, retinoic acid-induced cell death in the Hammertoe mutant resulted in correction of the mutant phenotype. Future studies will determine the relationship between exogenous agents and endogenous signaling pathways as well as indicate how these interactions can alter the fate of a given cell and potentially ameliorate a genetic abnormality.

Differential expression of clusterin in the testis and epididymis of postnatal and germ cell deficient mice.

Clusterin is found in a wide variety of tissues and is expressed in a number of physiological and pathological contexts. It is expressed constitutively in the adult male reproductive tract, specifically the testes and caput of the epididymis. The gene is also induced during apoptotic cell death in the kidney and hormone-dependent tissues such as the prostate and mammary gland. The mechanisms controlling the expression of clusterin gene expression in these tissues are still unknown, although it has been suggested that interactions between the Sertoli cells and germ cells in the testis, or epithelial cells and germ cells in the epididymis, may be required for clusterin expression. To investigate the importance of germ cells in the induction of clusterin expression in these tissues, we have used in situ hybridization to determine the developmental regulation and germ cell dependence of clusterin expression in the reproductive tract of the normal and germ cell deficient male mice. Clusterin mRNA is present in the supporting cells in the testes of normal mice and in both atrichosis (at) and dominant spotting mutant (Wv) mice, both of which are germ cell deficient. On the other hand, the expression of clusterin in the epididymis appears to be at least partially dependent of the presence of germ cells, because segmental localization of clusterin mRNA within the caput of the epididymis that is seen in normal mice is disrupted in the at and Wv mutants. In these germ cell deficient mutants, the steady-state level of clusterin mRNA, which is repressed in segment 1 and 2 of the adult wild type mouse, is expressed at levels similar to those seen at days 7 and 17 during the development of the normal epididymis.

Expression of clusterin in cell differentiation and cell death.

Clusterin, originally isolated as testosterone-repressed prostate message-2 from regressing rat ventral prostate, has been identified with the process of active cell death (ACD). The clusterin gene product is a glycosylated dimer consisting of alpha and beta subunits, resulting from the 70-kilodalton preprotein. To determine its relationship with ACD, we have examined clusterin expression via in situ hybridization and immunohistochemistry. Clusterin message is detected in the supporting cells in both testes and ovaries and the protein surrounds the mature germ cells. The highest level of expression was found in the head region of the epididymis. Clusterin message is also detected in selected cells of uterine glands and ducts both in the normal and pregnant uterus. The expression of clusterin in the developing embryo is most abundant in the choroid plexus, inner ear, and epithelium of the eye. None of the cells in the testes, epididymis, or embryo that express clusterin are undergoing ACD. The expression of clusterin appears to correlate with cell remodelling or differentiation that occurs during these periods of development. However, in the female reproductive system, we found clusterin to be expressed in both differentiating as well as dying cells. These results suggest that clusterin may provide support for cells undergoing specific biochemical and (or) physical changes. Our results are consistent with the hypothesis that clusterin is an antiinflammatory agent.

Apoptotic cell death in the limb and its relationship to pattern formation.

Detection of cell death throughout embryogenesis demonstrates its importance in the normal form and function of the organism. We have examined cellular death during normal limb development by use of markers that display the morphology of cell death, the presence of phagocytic cells, and lysosomal activity. In addition in situ labeling confirms fragmentation of DNA in the mammalian limb. By these criteria, cell death in the developing limb can be categorized as type 1 or apoptotic cell death. However, the signal(s) responsible for cellular destruction and activation of phagocytosis by neighboring cells or recruited macrophages remain to be identified. The decision for cellular fate during development and regulation of it once the decision is made are key questions. To address the specific question of what determines that one cell will die while its neighbor survives, we have used compounds, such as retinoic acid (RA), that have been shown to alter the pattern of normal development. We and others have shown that RA does indeed alter the pattern of cell death to the extent of inducing malformations in the limb. The mouse mutant Hammertoe (Hm) provides an abnormal system in which the pattern of cell death is specifically altered in the interdigital regions of the limb. Our preliminary data suggest that RA can also introduce cell death between digits 2, 3, 4, and 5 of the Hm mutant where there was no cell death to begin with. Our observations of the effect of RA on mutant limbs suggest that a direct relationship between RA and cell death does exist and that this interaction may be required for correct pattern formation. The alteration in the pattern of cell death in the mutant mouse is of great interest, since it would provide a rare example of specific correction of a birth defect by direct intercession against the physiological effect of the mutation.

Delayed internucleosomal DNA fragmentation in programmed cell death.

DNA fragmentation was evaluated in three instances of programmed cell death, interdigital cell death in embryonic mouse limbs, and metamorphic death of both the labial glands and intersegmental muscle in the tobacco hornworm Manduca sexta. In the mouse, we evaluated both developmental cell death and expanded-range cell death induced by retinoic acid. The status of DNA was examined in several ways. Nuclei were examined by electron microscopy and Feulgen staining. Quantitative assessment of total DNA content in Feulgen-stained degenerating nuclei was made for the gland. In the labial gland, DNA content does not drop during the early phases of cell death; nor is an endonucleolytic ladder seen when DNA was examined by ethidium bromide staining or prelabeling with [3H]thymidine. Only by using end labeling of DNA could we detect DNA fragmentation at a very late stage in cell death, day 4 of the collapse of the gland. In contrast, WEHI 7.1 lymphoma cells display an early and extensive ladder after treatment with glucocorticoids. In mouse limb, for which cell death follows a more classic apoptotic morphology, a ladder is likewise not seen. We conclude that activation of an endonuclease is neither a trigger nor a necessary or defining component of the early phases of developmental programmed cell death, and that reported failure by others to find such a ladder may depend on limitations in the system that is under investigation.

Stage and lineage-regulated expression of two hsp90 transcripts during mouse germ cell differentiation and embryogenesis.

The expression of members of the heat shock protein 90 (hsp90) gene family during testicular and embryonic development was investigated. Two different hsp90 transcripts were detected in RNA from mouse testis, approximately 3.2 kb and 2.9 kb in size, and were shown to exhibit cellular and developmental stage specificity of expression. The larger, more abundant transcript was expressed at high levels in the germinal compartment of the testis, particularly in germ cells in meiotic prophase. The smaller hsp90 transcript was expressed predominantly in the somatic compartment of the testis. Expression of the two hsp90 transcripts was observed in testes of other species, suggesting an important role for hsp90 in mammalian testicular function. In addition, expression of both hsp90 transcripts was detected in the embryonic and extra-embryonic compartments of mid-gestation embryos.

Characterization and inducibility of hsp 70 proteins in the male mouse germ line.

The properties and inducibility of the heat shock protein 70 (hsp 70) gene products were examined during differentiation of mouse testicular cells by one and two-dimensional gel electrophoresis and immunoblotting. Low levels of the 72- and 73-kD heat shock proteins normally found in mouse cell lines were detected in the mouse testis. A novel isoform with a relative molecular mass of 73 kD (called 73T) was also observed, in the presence or absence of heat shock. 73T was shown to be produced by germ cells since it was not detected in testes from mutant mice devoid of germ cells. Furthermore, 73T was found only in adult mouse testicular cells, not in testes from animals that lack meiotic germ cells. 73T was synthesized in enriched cell populations of both meiotic prophase and postmeiotic cells, but was not inducible by in vitro heat shock. In the adult testis, low levels of the bona fide 72-kD heat-inducible (hsp72) were induced in response to elevated temperatures. In contrast, in testes from animals in which only somatic cells and premeiotic germ cells were present, there was a substantial induction of hsp 72. It is suggested that hsp 72 is inducible in the somatic compartment and possibly in the premeiotic germ cells, but not in germ cells which have entered meiosis and which are expressing members of the hsp 70 gene family in a developmentally regulated fashion.

Programmed cell death: new thoughts and relevance to aging.

Cell death is a common phenomenon in developmental biology, and recent data suggest that it is as tightly regulated as mitosis. For numerous systems endocrine and neuronal factors are required to maintain viability of cells, as are specific diffusible and other unknown factors deriving from intimate cell-to-cell contact; and, in some instances, specific hormones or other circulating factors induce spontaneous self-destruction by the targeted cells. Some cells such as thymocytes may be primed to self-destruct and hence activate specific enzymes. In others, the doomed cell up-regulates a limited number of genes just before it dies. Of these genes, several are known but are not considered to cause cell death; others are under investigation. Although the situation is clearest for developmental biology, it appears that the presumptively random loss of cells in senescence results from invocation of the same mechanisms. Understanding and control of these mechanisms could conceivably lead either to protection against cell loss or specific induction of lysis in malignant cells.

Identification and sequence analysis of a new member of the mouse HSP70 gene family and characterization of its unique cellular and developmental pattern of expression in the male germ line.

A unique member of the mouse HSP70 gene family has been isolated and characterized with respect to its DNA sequence organization and expression. The gene contains extensive similarity to a heat shock-inducible HSP70 gene within the coding region but diverges in both 3' and 5' nontranslated regions. The gene does not yield transcripts in response to heat shock in mouse L cells. Rather, the gene appears to be activated uniquely in the male germ line. Analysis of RNA from different developmental stages and from enriched populations of spermatogenic cells revealed that this gene is expressed during the prophase stage of meiosis. A transcript different in size from the major heat-inducible mouse transcripts is most abundant in meiotic prophase spermatocytes and decreases in abundance in postmeiotic stages of spermatogenesis. This pattern of expression is distinct from that observed for another member of this gene family, which was previously shown to be expressed abundantly in postmeiotic germ cells. These observations suggest that specific HSP70 gene family members play distinct roles in the differentiation of the germ cell lineage in mammals.

Translational regulation of the novel haploid-specific transcripts for the c-abl proto-oncogene and a member of the 70 kDa heat-shock protein gene family in the male germ line.

Expression of the c-abl proto-oncogene in the mouse testis is characterized by the production of a unique 4.7-kb transcript present in germ cells that have entered the haploid phase of spermatogenesis. A similar developmental stage specificity of expression is observed for a member of the 70-kDa heat-shock protein (hsp 70) gene family. A unique-sized hsp 70 transcript (T-hsp 70) is produced in haploid spermatids and is stable throughout spermatogenesis. In the present study, we examined the regulation of expression of these genes by examining their association with polyribosomes. The germ cell-specific c-abl and T-hsp 70 mRNA variants were both associated with the polysomal fractions of mouse testis cells, suggesting that they are functional mRNAs. However, both c-abl and T-hsp 70 mRNAs were also found in the ribonucleoprotein particle fractions. The distribution of these mRNAs in both the polysomal and nonpolysomal fractions is comparable to that seen for the mRNA of protamine-1, a gene whose expression in the testis is known to be regulated at the level of translation. In contrast, transcripts from the beta-tubulin gene were seen predominantly in the polyribosomal fractions. These findings suggest that translation of the novel c-abl and T-hsp 70 transcripts is confined to subpopulations of testicular cells.

Developmental-stage-specific expression of the hsp70 gene family during differentiation of the mammalian male germ line.

Mouse somatic tissues contain low levels of transcripts homologous to the heat shock-inducible and cognate members of the heat shock protein 70 (hsp70) gene family. An abundant, unique sized hsp70 mRNA of 2.7 kilobases (kb) is present in testes in the absence of exogenous stress. Its expression is restricted to germ cells and is developmentally regulated. The 2.7-kb transcript first appears during the haploid phase of spermatogenesis and is stable throughout the morphogenic stages of spermiogenesis. A 2.7-kb hsp70 mRNA is present in rat and human testes. These observations suggest that a member of the hsp70 gene family plays a role in the development of the mammalian male germ cell lineage.

Differential expression of the c-abl proto-oncogene and the homeo box-containing gene Hox 1.4 during mouse spermatogenesis.

Mammalian spermatogenesis is a complex developmental process. Spermatozoa, like ova, are uniquely capable of supporting embryonic development. Our approach to understanding this process is to identify genes whose developmental pattern of expression suggests that they may play a role in spermatogenesis. Experiments on the cellular oncogene c-abl and the homeo box-containing gene Hox-1.4 indicate that these genes may be important for male germ cell development. Both genes produce testis-specific transcripts that are present in particular cellular populations of the adult testis. Their developmental specificity, however, is different: c-abl is haploid-specific, whereas Hox-1.4 is expressed in the germ cells as soon as they have entered meiosis. Future studies will focus on examining the protein products of these genes and their function in testicular cells.

Isolation of a mouse cDNA coding for a developmentally regulated, testis-specific transcript containing homeo box homology.

A clone, pHBT-1, containing sequences homologous to Drosophila homeo boxes has been isolated from a mouse testis cDNA library. The sequence is 80% homologous at the DNA level and 88% homologous at the amino acid level to the homeo box sequence of the Antennapedia gene of Drosophila. Sequences flanking the 3' end of the homeo box are highly diverged from other murine homeo box-containing genes characterized to date. RNA blot hybridization analysis of mouse testis poly(A)+ RNA revealed transcripts of approximately 1.4 kb in length. Within the limits of sensitivity of detection of Northern blot analysis, no transcripts were seen in any of the adult somatic tissues examined. Other tissues that contain stem cells, namely those of the hemopoietic system, also lacked detectable amounts of HBT-1 transcripts. HBT-1 transcripts were limited to male germ cell-containing tissues, since RNAs from juvenile and adult ovaries did not contain detectable amounts of the 1.4-kb transcripts. Expression of the HBT-1 gene was not detected in embryonic testes, nor in tests of neonatal animals which contain germ cells up to the Type B stage of spermatogonial development. A role for the expression of the HBT-1 gene in the meiotic stages of male germ cell differentiation is postulated.