[What makes a parasite "transforming"? Insights into cancer from the agents of an exotic pathology, Theileria spp]. / Comment et pourquoi un parasite peut-il être « transformant ¼ ? Apports d'agents de zoonoses exotiques, Theileria spp., à l'étude du cancer.

Theileria are obligate eukaryotic intracellular parasites of cattle. The diseases they cause, Tropical theileriosis and East Coast Fever, cause huge economic loss in East African, Mediterranean and central and South-East Asian countries. These apicomplexan parasites are the only intracellular eukaryotic parasites known to transform their host cell and represent a unique model to study host-parasite interactions and mechanisms of cancer onset.Here, we review how Theileria parasites induce transformation of their leukocyte host cell and discuss similarities with tumorigenesis. We describe how genomic innovation, epigenetic changes and hijacking of signal transductions enable a eukaryotic parasite to transform its host cell.

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Theileria induces oxidative stress and HIF1α activation that are essential for host leukocyte transformation.

Complex links between infection and cancer suggest that we still can learn much about tumorigenesis by studying how infectious agents hijack the host cell machinery. We studied the effects of an intracellular parasite called Theileria that infects bovine leukocytes and turns them into invasive cancer-like cells. We investigated the host cells pathways that are deregulated in infected leukocytes and might link infection and lymphoproliferative disease. We show that intracellular Theileria parasites drive a Warburg-like phenotype in infected host leukocytes, characterized by increased expression of metabolic regulators, increased glucose uptake and elevated lactate production, which were lost when the parasite was eliminated. The cohabitation of the parasites within the host cells leads to disruption of the redox balance (as measured by reduced/oxidized glutathione ratio) and elevated ROS (reactive oxygen species) levels, associated with chronic stabilization of the hypoxia-inducible factor 1 alpha (HIF1α). Inhibition of HIF1α (pharmacologically or genetically), or treatment with antioxidants, led to a marked reduction in expression of aerobic glycolytic genes and inhibited the transformed phenotype. These data show that stabilization of HIF1α, following increased ROS production, modulates host glucose metabolism and is critical for parasite-induced transformation. Our study expands knowledge about the molecular strategy used by the parasite Theileria to induce the transformed phenotypes of infected cells via reprogramming of glucose metabolism and redox signaling.

A role for AP-1 in apoptosis: the case for and against.

The nuclear transcription factor AP-1, composed of dimers of Fos and Jun proteins, has been linked to a startling breadth of cellular events including cell transformation, proliferation, differentiation and apoptosis. AP-1 is often portrayed as a general, nuclear decision-maker that determines life or death cell fates in response to extracellular stimuli. However, it is increasingly clear that the cellular context is critical for determining the contribution of AP-1 to cellular fates, and the role of AP-1 in apoptosis should be considered within the context of a complex network of nuclear factors that respond simultaneously to a wide range of signal transduction pathways. We take a closer look at the evidence for and against a role for AP-1 in inducing apoptosis, drawing on examples of studies in neurons, lymphocytes and hepatocytes. Although AP-1 activation is associated with a large number of apoptotic scenarios, its role in ensuring cell survival seems equally important. It is, therefore, difficult to convict AP-1 as a killer without taking into account the cellular and extracellular context within which it is functioning. Defining the target genes regulated by AP-1 in these different contexts will help to decipher the contribution of AP-1 to cell fate decisions.

Life and death in the JUNgle.

Experiments with transgenic and knockout mice have begun to elucidate distinct roles for the three members of the Jun family of transcription factors. Mice with tissue-specific loss of JunB develop a myeloproliferative disorder, emphasizing the important roles that Jun proteins play in regulating life and death decisions in disease.

JunD protects cells from p53-dependent senescence and apoptosis.

JunD is the most broadly expressed member of the Jun family and the AP-1 transcription factor complex. Primary fibroblasts lacking JunD displayed p53-dependent growth arrest, upregulated p19(Arf) expression, and premature senescence. In contrast, immortalized cell lines lacking JunD showed increased proliferation and higher cyclinD1 levels. These properties are reminiscent of the effects of oncogenic Ras expression on primary and established cell cultures. Furthermore, JunD(-/-) fibroblasts exhibited increased p53-dependent apoptosis upon ultraviolet irradiation and were sensitive to the cytotoxic effects of TNF-alpha. The antiapoptotic role of JunD was confirmed using an in vivo model of TNF-mediated hepatitis. We propose that JunD protects cells from senescence, or apoptotic responses to stress stimuli, by acting as a modulator of the signaling pathways that link Ras to p53.

Targeted disruption of the murine junD gene results in multiple defects in male reproductive function.

JunD is one of three mammalian Jun proteins that contribute to the AP-1 transcription factor complex. Distinct regulation and functions have been proposed for each Jun member, but less is known about the biological functions of each of these proteins in vivo. To investigate the role of JunD, we have inactivated the murine gene by replacement with a bacterial lacZ reporter gene. Embryonic JunD expression was initially detected in the developing heart and cardiovascular system. Subsequent broadening phases of JunD expression were observed during embryonic development and expression in the adult was widespread in many tissues and cell lineages. Mutant animals lack JunD mRNA and protein and showed no evidence of upregulation of c-Jun and JunB mRNA levels. In contrast to the other two Jun members, homozygous JunD-/- mutant animals were viable and appeared healthy. However, homozygous JunD-/- animals showed a reduced postnatal growth. Furthermore, JunD-/- males exhibited multiple age-dependent defects in reproduction, hormone imbalance and impaired spermatogenesis with abnormalities in head and flagellum sperm structures. No defects in fertility were observed in JunD-/- female animals. These results provide evidence for redundant functions for members of the Jun family during development and specific functions for JunD in male reproductive function.

Examining the relationship between the gelatinolytic balance and the invasive capacity of endothelial cells.

Angiogenesis and the formation of new blood vessels requires coordinated regulation of matrix proteolysis and endothelial cell migration. Cellular proteolytic capacity is the balance between secreted matrix metalloproteinases (MMP) and their inhibitors (TIMPs). We have examined the regulation of the gelatinase/TIMP balance by transforming growth factor-beta1 (TGF-beta1) and phorbol myristate acetate (PMA) in bovine endothelial cells. The low constitutive expression of gelatinase A/MMP-2 was upregulated by TGF-beta1 in a dose-dependent manner. Gelatinase B/MMP-9 was only detected upon treatment with either PMA or TGF-beta1. However, addition of both factors together revealed a striking synergistic effect causing upregulation of MMP-9 and downregulation of TIMPs, thereby increasing the net MMP-9/TIMP balance and the gelatinolytic capacity. These effects were observed at both the protein and mRNA levels. We demonstrate that changes in different members of the Jun oncogene family with distinct transactivation properties may account for this synergistic effect. We investigated the contribution of these changes in gelatinolytic balance to endothelial cell migration and invasion. The endothelial cells showed increased cell motility in response to PMA, but the addition of TGF-beta1 had an inhibitory effect. Hence, regulation of the MMP-9/TIMP balance failed to correlate with the migratory or invasive capacity. These results question a direct role for MMP-9 in endothelial cell motility and suggest that gelatinases may contribute in alternative ways to the angiogenic process.

Signal transduction pathways and modulation of gene activity.

Research over the last decade has provided us with much information about the mechanisms that integrate signal transduction pathways with specific gene expression programs. We discuss the types of mechanisms used by different pathways to modulate transcription factor activity, citing examples from diverse molecular systems. Careful regulation of these pathways is essential to maintain balanced transcriptional control. Understanding the mechanisms that control gene activity will enable us to intervene therapeutically in diseases associated with deregulated transcriptional activity.

Medical examiner asthma death autopsies: a distinct subgroup of asthma deaths with implications for public health preventive strategies.

OBJECTIVE: Asthma deaths have been increasing in the United States and worldwide. We studied medical examiner asthma death autopsy (MEADA) records for the state of Maryland, compared selected characteristics with state and national total asthma deaths (TADs), and comprehensively reviewed relevant literature to define characteristics of asthma deaths and to provide insight for the design of future preventive strategies directed at this subgroup. DESIGN: Protocols for autopsy and clinical data. SETTING: The Office of the Chief Medical Examiner of the State of Maryland. SUBJECTS: All MEADAs in the state of Maryland from 1988 through 1992. MAIN OUTCOME MEASURES: Descriptive analysis. RESULTS: Maryland MEADAs (63 cases) represented 16.62% of Maryland TADs (379 cases). Most common characteristics of individuals on whom autopsies were performed: inner-city residence; single; black male; 15 to 54 years old; history of asthma; no other significant medical condition; fatal episode more likely to begin at home; pronounced dead at hospital; time of death between midnight and 6 AM; no particular seasonality; and typical gross and/or microscopic pathology. Analysis also revealed that 17.46% of deceased asthma patients had a history of drug abuse; 12.69% had positive toxicology for drugs of abuse; 9.52% were infants and young children up to 4 years old, all of whom were found, unresponsive, at home; and white females comprised the highest number of TADs but the lowest number of MEADAs. CONCLUSION: Asthma education programs focused on asthmatic inner-city black males, especially those with a history of drug abuse, and on parents of inner-city asthmatic infants and children may be a useful preventive strategy. International, national, and regional MEADA databases may also be of use in the design and monitoring of preventive strategies directed at this subgroup.

Integrin alpha chain cytoplasmic tails regulate "antibody-redirected" cell adhesion, independently of ligand binding.

Here we describe a novel "antibody-redirected cell adhesion" (ARCA) assay. This assay measures heterotypic cell-cell adhesion, resulting from antibody bridging between Fc gamma receptors type II (CD32) on leukocytes, and clustered integrins on adherent cell monolayers. This ARCA activity, facilitated by integrins alpha3 beta1 or alpha4 beta1, required an intact cytoskeleton, but did not involve typical integrin ligand binding sites or divalent cations. Furthermore, deletion of the alpha4 cytoplasmic tail almost completely abrogated integrin ARCA activity, suggesting an alteration of integrin recruitment into adhesive sites. If two or more tail residues were present after the conserved GFFKR motif, then ARCA activity was largely restored. Although alpha4 tail deletion caused loss of ARCA activity, it had no effect on the binding of VCAM-1 to intact alpha4-transfected K562 cells. In conclusion, the integrin alpha chain tail can positively regulate integrin-dependent cell adhesion by a receptor recruitment/clustering mechanism independent of conventional integrin ligand-binding considerations.

Ambulatory monitoring devices and the forensic autopsy.

Ambulatory monitoring devices (AMDs) such as Holter (ECG) and apnea (respiratory) monitors with built-in date- and time-correlated memories are occasionally encountered in the forensic autopsy. Diagnostic data are usually readily obtained by returning the device to the hospital department or rental company. This data can be easily correlated with autopsy findings to arrive at surprisingly precise conclusions in some cases. We present two illustrative cases: an elderly man wearing a Holter monitor who shot himself in the mouth with a shotgun, and a 15-month-old oxygen-dependent prematurely born boy with bronchopulmonary dysplasia whose apnea monitor had been turned off 5 days before he died of bronchopneumonia. We discuss other kinds of AMDs that may be encountered in the forensic autopsy and suggest that information from these monitors perhaps should become part of the forensic autopsy report.

Reduction of tumorigenicity by alpha 3 integrin in a rhabdomyosarcoma cell line.

The expression levels of integrin adhesion receptors have often been correlated with neoplastic transformation and invasiveness. To investigate more definitively the role of the integrin VLA-3 (alpha 3 beta 1) in tumor cell behavior, we transfected alpha 3 subunit cDNA into human rhabdomyosarcoma (RD) cells. Transfectants expressing high levels of alpha 3 beta 1 on their cell surface displayed an altered morphology and decreased anchorage-dependent growth in vitro. Cells expressing alpha 3 also displayed marked reduction in anchorage-independent growth in soft agar and in their ability to form tumors when injected subcutaneously into athymic nude mice. Thus, VLA-3 can repress the transformed phenotype of rhabdomyosarcoma tumor cells. Similar changes in morphology and growth characteristics were observed in cells expressing a chimeric molecule X3C4 in which the alpha 3 cytoplasmic domain had been exchanged with that of the alpha 4 integrin subunit. Therefore, alpha 3 inhibitory effects in RD cells appear not to require specific signalling through the alpha 3 cytoplasmic domain.