The field of cancer research: an indicator of present transformations in biology.

'Biology and cancer research have developed together. Invariably, at each stage, the characteristics of the cancer cell have been ascribed to some defect in whatever branch of biology happens at the time to be fashionable and exciting; today, it is molecular genetics'. Tremendous transformations have occurred in cancer research since these few lines were written by John Cairns: the discovery of oncogenes and anti-oncogenes, and the successful development of 'magic bullets' targeting the proteins encoded by these oncogenes. Nevertheless, Cairns' message is still valid. In 1978, he observed the first attempts to apply the tools and concepts of molecular biology to cancer; today, this research field reflects multiple and diverse efforts that go 'beyond' molecular biology by looking for explanations that have been left aside during its development, or by privileging new approaches, fully original or actively pursued in other fields of biological research. Because of this specific characteristic of cancer research, it is possible to use it as an indicator of trends in biological research in general.

HSFs in development.

Heat shock transcription factors, as well as heat shock proteins, are involved in different steps in differentiation and development, in addition to their role in adaptation to stress. This has already been demonstrated in the case of the single heat shock factor present in Drosophila. Over the last 6 years, similar observations have accumulated from the progressive inactivation of the different hsf genes in mammals, the use of double-null animals, and the slow characterization of their complex phenotypes. Although these studies are not yet complete, the data so far can be used to draw some conclusions. All hsf genes contribute to development in mammals and to normal functions at the adult stage, by controlling the expression of Hsp and non-Hsp genes. Reproduction, the immune response and aging are the processes that are the most deeply affected. An attractive hypothesis would be that these new functions have been recruited during evolution in order to coordinate these processes: HSFs may occupy a central place in the trade off that organisms make between reproduction and maintenance, in response to the variations in the environment.

Henri Atlan's early writings.

The importance given to biological complexity and dynamics, the progressive giving up the notion of genetic programme, the attention paid to noise at the molecular level, are all new aspects of biological research characteristic of the post-genomic era. They are frequently mentioned as the signs of the delayed recognition of the important contributions made by Henri Atlan from the beginning of the 1970s. By focusing my study on his first major essay, l'Organisation Biologique et la Théorie de l'Information, I reach a somewhat different conclusion. Whereas Henri Atlan proposed an early criticism of the notion of genetic programme, the point of view he adopted was at odds with molecular biology. His attempt to apply the theory of information to organisms was a project already abandoned in the United States. The importance he placed to the second law of thermodynamics and his insistence on the limits of the Darwinian theory of evolution were more the sign of an attachment to past traditions of biological thought, still permeating the French community of biologists in the second half of the XXth century, than an anticipation of future developments in the post-genomic era.

Phenotypic characterization of mouse embryonic fibroblasts lacking heat shock factor 2.

In murine cells, the heat shock response is regulated by a transcription factor, HSF1, which triggers the transcription of heat shock genes. HSF2 has been shown to be involved in meiosis and mouse brain development. We characterized the effects of the absence of HSF2 in mouse embryonic fibroblasts (MEFs). The temperature threshold of the heat shock response appeared lowered in Hsf2(-/-) MEFS as monitored by the synthesis of heat shock protein HSP70. In contrast to unstressed wild type MEFS, HSP70 and HSF1 are localized in the nucleus of unstressed Hsf2(-/-) MEFS, a characteristic of stressed cells. HSF1 is not activated for DNA-binding at unstressed temperature in Hsf2(-/-) MEFS. Therefore, the absence of HSF2 induces some but not all of the characteristics of the stress response. In addition, Hsf2(-/-) MEFS exhibited proliferation defects, altered morphology, remodeling of the fibronectin network.

Overexpression of murine small heat shock protein HSP25 interferes with chondrocyte differentiation and decreases cell adhesion.

Although multiple functions for the small heat shock protein HSP25 have been proposed, its specific role during developmental and differentiation processes is not known. Cartilage is one of the tissues in which HSP25 is specifically and highly expressed during development. C1 cells, able to form aggregates in vitro, can be induced to differentiate into chondrocytes. In this study, we generated two stable transfected clones overexpressing HSP25 at two different levels. Cell morphology and growth rate were modified in both clones, although the actin content and distribution did not seem to be altered. Overexpressing clones had more difficulties in coalescing, leading to smaller aggregates and they did not differentiate into chondrocytes. Subsequently, these aggregates tended to dissociate into loose masses of dying cells. The strength of all these effects was directly correlated to the level of HSP25 overexpression. These data suggest that overexpressing HSP25 decreases cellular adhesion and interferes with chondrocyte differentiation.

The bacterial nucleoside N(6)-methyldeoxyadenosine induces the differentiation of mammalian tumor cells.

Contrary to bacterial DNA, mammalian DNA contains very little if any N(6)-methyldeoxyadenosine (MDA). The possible biological effect of this nucleoside on eukaryotic cells has been studied on different tumor cell lines. Addition of MDA to C6.9 glioma cells triggers a differentiation process and the expression of the oligodendroglial marker 2',3'-cyclic nucleotide 3'phosphorylase (CNP). The biological effects of N(6)-methyldeoxyadenosine were not restricted to C6.9 glioma cells since differentiation was also observed on pheochromocytoma and teratocarcinoma cell lines and on dysembryoplastic neuroepithelial tumor cells. The precise mechanism by which MDA induces cell differentiation remains unclear, but is related to cell cycle modifications. These data point out the potential interest of N(6)-methyldeoxyadenosine as a novel antitumoral and differentiation agent. They also raise the intriguing question of the loss of adenine methylation in mammalian DNA. Furthermore, the finding that a methylated nucleoside found in bacterial DNA induces a biological process might have implications in gene therapy approaches when plasmid DNAs are injected into humans.

The distribution of heat shock proteins in the nervous system of the unstressed mouse embryo suggests a role in neuronal and non-neuronal differentiation.

Heat shock proteins (Hsps) act as molecular chaperones and are generally constitutively expressed in the absence of stress. Hsps are also inducible by a variety of stressors whose effects could be disastrous on the brain. It has been shown previously that Hsps are differentially expressed in glial and neuronal cells, as well as in the different structures of the brain. This differential expression has been related to specific functions distinct from their general chaperone function, such as intracellular transport. We investigated here the constitutive expression of 5 Hsps (the small Hsp, Hsp25, the constitutive Hsc70 and Hsp90beta, the mainly inducible Hsp70 and Hsp90alpha), and of a molecular chaperone, TCP-1alpha during mouse nervous system development. We analyzed, by immunohistochemistry, their distribution in the central nervous system and in the ganglia of the peripheral nervous system from day 9.5 (E9.5) to day 17.5 (E17.5) of gestation. Hsps are expressed in different cell classes (neuronal, glial, and vascular). The different proteins display different but often overlapping patterns of expression in different regions of the developing nervous system, suggesting unique roles at different stages of neural maturation. Their putative function in cell remodeling during migration or differentiation and in protein transport is discussed. Moreover we consider Hsp90 function in cell signaling and the role of Hsp25 in apoptosis protection.

Heat shock and arsenite induce expression of the nonclassical class I histocompatibility HLA-G gene in tumor cell lines.

The nonclassical histocompatibility class I gene HLA-G has a tissue-restricted expression. To explore mechanisms involved in HLA-G transcriptional regulation, we have investigated the effect of stress, including heat shock and arsenite treatment, on HLA-G expression in tumor cell lines. We show that stress induces an increase of the level of the different HLA-G alternative transcripts without affecting other MHC class I HLA-A, -B, -E, and -F transcripts. A heat shock element (HSE) that binds to heat shock factor 1 (HSF1) on stress conditions was further identified within the HLA-G promoter. Considering the ability of HLA-G to modulate the function of immunocompetent cells, we hypothesize a new feature of HLA-G as a signal regulating the immune response to stress.

Cyclosporin A induces an atypical heat shock response.

Cyclosporin A is a widely used immunosuppressive drug having toxic side effects, in particular on kidneys and liver, as a result of its action on different molecular targets. Here we demonstrate that low doses of CsA are able to induce the expression of the heat shock protein HSP27 and its hyperphosphorylation. It also activates the two heat shock transcription factors, HSF1 and HSF2. Since these factors have been shown to be activated by proteasome inhibition, we tested the hypothesis that the inhibitory action of CsA on the proteasome might be responsible for the activation of HSFs and the subsequent expression of HSP27. The increase in multiubiquitinated proteins as well as the stabilization of p53 following CsA addition argues in favor of this hypothesis. The kidney BSC-1 cells are highly responsive to the addition of CsA: the possible link between HSP27 induction and hyperphosphorylation and nephrotoxicity is discussed.

Hsp25 and the p38 MAPK pathway are involved in differentiation of cardiomyocytes.

The small heat-shock protein HSP25 is expressed in the heart early during development, and although multiple roles for HSP25 have been proposed, its specific role during development and differentiation is not known. P19 is an embryonal carcinoma cell line which can be induced to differentiate in vitro into either cardiomyocytes or neurons. We have used P19 to examine the role of HSP25 in differentiation. We found that HSP25 expression is strongly increased in P19 cardiomyocytes. Antisense HSP25 expression reduced the extent of cardiomyocyte differentiation and resulted in reduced expression of cardiac actin and the intermediate filament desmin and reduced level of cardiac mRNAs. Thus, HSP25 is necessary for differentiation of P19 into cardiomyocytes. In contrast, P19 neurons did not express HSP25 and antisense HSP25 expression had no effect on neuronal differentiation. The phosphorylation of HSP25 by the p38/SAPK2 pathway is known to be important for certain of its functions. Inhibition of this pathway by the specific inhibitor SB203580 prevented cardiomyocyte differentiation of P19 cells. In contrast, PD90589, which inhibits the ERK1/2 pathway, had no effect. Surprisingly, cardiogenesis was only sensitive to SB203580 during the first 2 days of differentiation, before HSP25 expression increases. In contrast to the effect of antisense HSP25, SB203580 reduced the level of expression of the mesodermal marker Brachyury-T during differentiation. Therefore, we propose that the p38 pathway acts on an essential target during early cardiogenesis. Once this initial step is complete, HSP25 is necessary for the functional differentiation of P19 cardiomyocytes, but its phosphorylation by p38/SAPK2 is not required.

Gene function.

The problem of gene function--of the relationships between hereditary material and the characteristics of organisms--preceded the rediscovery of Mendel's laws and accompanied the development of genetics in the XXth century. Molecular biologists replaced the simple gene-character relationship by two relationships: the first, between genes and proteins, was well defined, whereas the second between proteins and the complex structural and functional characteristics of organisms remained unknown. I will describe in this article the experimental approaches which helped to characterize during the last twenty years the relationships between proteins and characters. Four principles of macromolecular organization emerged from these studies: conservation of the elementary components during evolution, existence of pathways and networks, pleiotropy and redundancy. These principles are the explanation of the surprising experimental observations that have been made in recent years. The existence of these principles makes problematic any prediction on the consequences of gene modification. It both sounds the death-knell of the simplistic reductionist approach of many biologists, whereby genes were considered as responsible for specific functions, and definitely prevents the distribution of genes in separate, well defined categories.

François Jacob's lab in the seventies: the T-complex and the mouse developmental genetic program.

The existence of a genetic program of development was proposed by molecular biologists in the nineteen-sixties. Historians and philosophers of science have since thoroughly criticized this notion. To fully appreciate its significance, it is interesting to consider the research which was pursued during this period by molecular biologists who proposed this notion. This study focuses on François Jacob's work and on the model of development supported by his lab in the early seventies, the T-complex model. This episode of Jacob's scientific activity has since been forgotten. Characterization of this model shows that the notion of program was used in a metaphoric way and that it did not put any constraint on the work pursued in the lab at that time. Some attention is devoted to the origin of this metaphor in the context of the nineteen-seventies.

Genomic structure and chromosomal localization of the mouse Hsf2 gene and promoter sequences.

The mouse heat shock factor 2 (HSF2) cDNA was previously cloned by homology to HSF1, the heat shock factor involved in the cellular response to stress [Sarge, K.D., Zimarino, V., Holm, K., Wu, C., Morimoto, R.I., Cloning and characterization of two mouse heat shock factors with distinct inducible and constitutive DNA-binding ability. Genes Dev. 5 (1991) 1902-1911]. HSF2 is active in restricted cell types during pre- and post-implantation stages of development, and only in male germ cells of adult mice. However, the function of this factor remains elusive. We report here the cloning of the mouse Hsf2 gene and its genomic structure. We show that the gene is composed of 13 exons of variable sizes spanning at least 43kb in the genome. The transcription start site has been determined, and upstream sequences with promoter activity have been identified by their ability to direct the expression of a luciferase reporter gene in transfected cells. A preliminary analysis of the proximal promoter sequence determined that the TATA box is absent, but that a GC-rich region with several potential binding sites for transcription factors is present. The gene has been mapped to mouse chromosome 10 by in-situ hybridization on metaphase chromosomes.

[Temporal regulation of gene expression]. / Régulation dans le temps de l'expression des gènes.

Molecular biology gives a static--not a dynamic--vision of the mechanisms regulating gene expression. Genetics already gave to time a limited place in the explanation of living phenomena. Such a static vision is supported by the techniques--such as X-ray crystallography--used by the biologists. However time is an important parameter in the control of gene expression during the cellular response to external signals, during life and aging of organisms or even in the succession of living forms which takes place in evolution. Models are slowly moving, due to the eruption of new technologies giving access to the fast events which occur inside living cells. A new dynamic vision is progressively replacing the old one. The consequences of these changes on the form of the future biology remain still unknown.