High incidence of lung, bone, and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene.

We have investigated the role of the p53 gene in oncogenesis in vivo by generating transgenic mice carrying murine p53 genomic fragments isolated from a mouse Friend erythroleukemia cell line or BALB/c mouse liver DNA. Elevated levels of p53 mRNA were detected in several tissues of two transgenic lines tested. Increased levels of p53 protein were also detected in most of the tissues analyzed by Western blotting (immunoblotting). Because both transgenes encoded p53 proteins that were antigenically distinct from wild-type p53, it was possible to demonstrate that overexpression of the p53 protein was mostly, if not entirely, due to the expression of the transgenes. Neoplasms developed in 20% of the transgenic mice, with a high incidence of lung adenocarcinomas, osteosarcomas, and lymphomas. Tissues such as ovaries that expressed the transgene at high levels were not at higher risk of malignant transformation than tissues expressing p53 protein at much lower levels. The long latent period and low penetrance suggest that overexpression of p53 alone is not sufficient to induce malignancies and that additional events are required. These observations provide direct evidence that mutant alleles of the p53 oncogene have oncogenic potential in vivo and that different cell types show intrinsic differences in susceptibility to malignant transformation by p53. Since recent data suggest that p53 may be a recessive oncogene, it is possible that the elevated tumor incidence results from functional inactivation of endogenous p53 by overexpression of the mutant transgene. The high incidence of lung and bone tumors suggests that p53 transgenic mice may provide a useful model to investigate the molecular events that underlie these malignancies in humans.     

Sequential phsophorylation of histone subfractions in the Chinese hamster cell cycle.

Ion exchange chromatography and preparative electrophoresis were used to examine the phosphorylation of histone f1 and f3 subfractions in synchronized Chinese hamster cells (line CHO). Three discrete f1 phosphorylation events were demonstrated to occur in sequence during the cell cycle. The first event (f1G1) commenced in G1 2 hours prior to entry of cells into S phase; the second event (f1s) commenced simultaneously with initiation of DNA synthesis; and the third event (f1M) commenced when cells entered mitosis. F1M phosphorylation occurred simultaneously with the phosphorylation of histone f3 (which is not phosphorylated during G1, S, or G2). Fractionation of f1 and f3 revealed no differences in these sequential phosphorylation patterns among the various f1 and f3 subfractions, indicating that these phosphorylations are general biochemical events of the cell cycle. Phosphorylated (f1G1) was found to accumulate in cells as they traversed THEIR CELL CYCLE. F1s was phosphorylated to twice the extent of f1G1, but f1s did not accumulate in the cells as they passed through interphase. F1M was phosphorylated to about 4 times the extent of the first phosphorylated form (f1G1). A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests that (a) f1G1 phosphorylation is involved with chromatin structural changes necessary for cell proliferation; (b) f1s phosphorylation is involved with DNA replication; (c) F1M and f3 phosphorylations are involved in chromosome condensation.     

IQGAP and mitotic exit network (MEN) proteins are required for cytokinesis and re-polarization of the actin cytoskeleton in the budding yeast, Saccharomyces cerevisiae

In budding yeast the final stages of the cell division cycle, cytokinesis and cell separation, are distinct events that require to be coupled, both together and with mitotic exit. Here we demonstrate that mutations in genes of the mitotic exit network (MEN) prevent cell separation and are synthetically lethal in combination with both cytokinesis and septation defective mutations. Analysis of the synthetic lethal phenotypes reveals that Iqg1p functions in combination with the MEN components, Tem1p, Cdc15p Dbf20p and Dbf2p to govern the re-polarization of the actin cytoskeleton to either side of the bud neck. In addition phosphorylation of the conserved PCH protein, Hof1p, is dependent upon these activities and requires actin ring assembly. Recruitment of Dbf2p to the bud neck is dependent upon actin ring assembly and correlates with Hof1p phosphorylation. Failure to phosphorylate Hof1p results in the increased stability of the protein and its persistence at the bud neck. These data establish a mechanistic dependency of cell separation upon an intermediate step requiring actomyosin ring assembly.     

A predictive model of cell traction forces based on cell geometry

Recent work has indicated that the shape and size of a cell can influence how a cell spreads, develops focal adhesions, and exerts forces on the substrate. However, it is unclear how cell shape regulates these events. Here we present a computational model that uses cell shape to predict the magnitude and direction of forces generated by cells. The predicted results are compared to experimentally measured traction forces, and show that the model can predict traction force direction, relative magnitude, and force distribution within the cell using only cell shape as an input. Analysis of the model shows that the magnitude and direction of the traction force at a given point is proportional to the first moment of area about that point in the cell, suggesting that contractile forces within the cell act on the entire cytoskeletal network as a single cohesive unit. Through this model, we demonstrate that intrinsic properties of cell shape can facilitate changes in traction force patterns, independently of heterogeneous mechanical properties or signaling events within the cell.     

Effects of the substratum on the migration of primordial germ cells

It is now clear from work on defined cell types on artificial substrates that various chemical and physical inhomogeneities in the substrates can guide cell locomotion. It is also becoming clear that less well defined inhomogeneities in living cell substrates can guide the normal locomotion of embryonic migratory cells in vivo. The primordial germ cells (p.g.cs) of early anuran amphibian embryos are proving a useful model for the study of cell migration. When isolated from the embryo and cultured on living cellular substrate, p.g.cs become oriented by the shapes of the underlying cells or by their stress fibre cytoskeleton, or both. A combination of scanning and transmission electron microscopy in vivo shows a clearly aligned cellular substrate for p.g.c. migration along part of their route. Furthermore, we find that the glycoprotein fibronectin is involved in p.g.c. adhesion, which suggests a link between orientation of the substrate cells and p.g.c. guidance.     

Early and Late Gene Changes in MPTP Mice Model of Parkinson's Disease Employing cDNA Microarray

Recently, we reported specific brain gene expression changes in the chronic MPTP model in the late stage of degeneration, employing cDNA expression array, which indicate a domino cascade of events involved in neuronal cell death. In an attempt to elucidate early gene expression profile in the region of the substantia nigra (SN) and the striatum of acute MPTP-treated mice (3–24 h), we elected a restricted number of genes affected by the long-term MPTP treatment, and their expression was examined. Specifically, we detected alterations in the expression of genes implicated in oxidative-stress, inflammatory processes, signal transduction and glutamate toxicity. These pro-toxic genes appear to be compensated by the elevated expression in trophic factors and antioxidant defenses, which are also activated by short exposure to MPTP. The time course of these gene expression changes indicates the importance of investigating the early gene cascade of events occurring prior to late nigrostriatal dopamine neuronal cell death.     

Monitoring single-channel water permeability in polarized cells

So far the determination of unitary permeability (p(f)) of water channels that are expressed in polarized cells is subject to large errors because the opening of a single water channel does not noticeably increase the water permeability of a membrane patch above the background. That is, in contrast to the patch clamp technique, where the single ion channel conductance may be derived from a single experiment, two experiments separated in time and/or space are required to obtain the single-channel water permeability p(f) as a function of the incremental water permeability (P(f,c)) and the number (n) of water channels that contributed to P(f,c). Although the unitary conductance of ion channels is measured in the native environment of the channel, p(f) is so far derived from reconstituted channels or channels expressed in oocytes. To determine the p(f) of channels from live epithelial monolayers, we exploit the fact that osmotic volume flow alters the concentration of aqueous reporter dyes adjacent to the epithelia. We measure these changes by fluorescence correlation spectroscopy, which allows the calculation of both P(f,c) and osmolyte dilution within the unstirred layer. Shifting the focus of the laser from the aqueous solution to the apical and basolateral membranes allowed the FCS-based determination of n. Here we validate the new technique by determining the p(f) of aquaporin 5 in Madin-Darby canine kidney cell monolayers. Because inhibition and subsequent activity rescue are monitored on the same sample, drug effects on exocytosis or endocytosis can be dissected from those on p(f).     

Gene Conversion and Functional Divergence in the β-Globin Gene Family

Different models of gene family evolution have been proposed to explain the mechanism whereby gene copies created by gene duplications are maintained and diverge in function. Ohta proposed a model which predicts a burst of nonsynonymous substitutions following gene duplication and the preservation of duplicates through positive selection. An alternative model, the duplication–degeneration–complementation (DDC) model, does not explicitly require the action of positive Darwinian selection for the maintenance of duplicated gene copies, although purifying selection is assumed to continue to act on both copies. A potential outcome of the DDC model is heterogeneity in purifying selection among the gene copies, due to partitioning of subfunctions which complement each other. By using the d_N/d_S () rate ratio to measure selection pressure, we can distinguish between these two very different evolutionary scenarios. In this study we investigated these scenarios in the -globin family of genes, a textbook example of evolution by gene duplication. We assembled a comprehensive dataset of 72 vertebrate -globin sequences. The estimated phylogeny suggested multiple gene duplication and gene conversion events. By using different programs to detect recombination, we confirmed several cases of gene conversion and detected two new cases. We tested evolutionary scenarios derived from Ohtas model and the DDC model by examining selective pressures along lineages in a phylogeny of -globin genes in eutherian mammals. We did not find significant evidence for an increase in the ratio following major duplication events in this family. However, one exception to this pattern was the duplication of -globin in simian primates, after which a few sites were identified to be under positive selection. Overall, our results suggest that following gene duplications, paralogous copies of -globin genes evolved under a nonepisodic process of functional divergence.[Reviewing Editor: Martin Kreitman]     

Genetic tumor archeology: microdissection and genetic heterogeneity in squamous and basal cell carcinoma

Carcinogenesis is a multi-step series of somatic genetic events. The complexity of this multi-hit process makes it difficult to determine each single event and the definitive outcome of such events. To investigate the genetic alterations in cancer-related genes, sensitive and reliable detection methods are of major importance for generating relevant results. Another critical issue is the quality of starting material which largely affects the outcome of the analysis. Microdissection of cells defined under the microscope ensures a selection of representative material for subsequent genetic analysis. Skin cancer provides an advantageous model for studying the development of cancer. Detectable lesions occur early during tumor progression, facilitating molecular analysis of the cell populations from both preneoplastic and neoplastic lesions. Alterations of the p53 tumor suppressor gene are very common in non-melanoma skin cancer, and dysregulation of p53 pathways appear to be an early event in the tumor development. A high frequency of epidermal p53 clones has been detected in chronically sun-exposed skin. The abundance of clones containing p53 mutated keratinocytes adjacent to basal cell (BCC) and squamous cell carcinoma (SCC) suggests a role in human skin carcinogenesis. Studies using p53 mutations as a clonality marker have suggested a direct link between actinic keratosis, SCC in situ and invasive SCC. Microdissection-based studies have also shown that different parts of individual BCC tumors can share a common p53 mutation yet differ with respect to additional alterations within the p53 gene, consistent with subclonal development within tumors. Here, we present examples of using well-defined cell populations, including single cells, from complex tissue in combination with molecular tools to reveal features involved in skin carcinogenesis.     

Binding and reversible denaturation of double-stranded DNA by Ff gene 5 protein

The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+).     

Hyaluronan-mediated CD44 interaction with RhoGEF and Rho kinase promotes Grb2-associated binder-1 phosphorylation and phosphatidylinositol 3-kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression

In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, alpha, beta, gamma forms but not the delta form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1.PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1.PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression.     

Cell cycle in the fucus zygote parallels a somatic cell cycle but displays a unique translational regulation of cyclin-dependent kinases

In eukaryotic cells, the basic machinery of cell cycle control is highly conserved. In particular, many cellular events during cell cycle progression are controlled by cyclin-dependent kinases (CDKs). The cell cycle in animal early embryos, however, differs substantially from that of somatic cells or yeasts. For example, cell cycle checkpoints that ensure that the sequence of cell cycle events is correct have been described in somatic cells and yeasts but are largely absent in embryonic cells. Furthermore, the regulation of CDKs is substantially different in the embryonic and somatic cells. In this study, we address the nature of the first cell cycle in the brown alga Fucus, which is evolutionarily distant from the model systems classically used for cell cycle studies in embryos. This cycle consists of well-defined G1, S, G2, and M phases. The purine derivative olomoucine inhibited CDKs activity in vivo and in vitro and induced different cell cycle arrests, including at the G1/S transition, suggesting that, as in somatic cells, CDKs tightly control cell cycle progression. The cell cycle of Fucus zygotes presented the other main features of a somatic cell cycle, such as a functional spindle assembly checkpoint that targets CDKs and the regulation of the early synthesis of two PSTAIRE CDKs, p32 and p34, and the associated histone H1 kinase activity as well as the regulation of CDKs by tyrosine phosphorylation. Surprisingly, the synthesis after fertilization of p32 and p34 was translationally regulated, a regulation not described previously for CDKs. Finally, our results suggest that the activation of mitotic CDKs relies on an autocatalytic amplification mechanism.     

Investigating the role of islet cytoarchitecture in its oscillation using a new beta-cell cluster model

The oscillatory insulin release is fundamental to normal glycemic control. The basis of the oscillation is the intercellular coupling and bursting synchronization of beta cells in each islet. The functional role of islet beta cell mass organization with respect to its oscillatory bursting is not well understood. This is of special interest in view of the recent finding of islet cytoarchitectural differences between human and animal models. In this study we developed a new hexagonal closest packing (HCP) cell cluster model. The model captures more accurately the real islet cell organization than the simple cubic packing (SCP) cluster that is conventionally used. Using our new model we investigated the functional characteristics of beta-cell clusters, including the fraction of cells able to burst f(b), the synchronization index lambda of the bursting beta cells, the bursting period T(b), the plateau fraction p(f), and the amplitude of intracellular calcium oscillation [Ca]. We determined their dependence on cluster architectural parameters including number of cells n(beta), number of inter-beta cell couplings of each beta cell n(c), and the coupling strength g(c). We found that at low values of n(beta), n(c) and g(c), the oscillation regularity improves with their increasing values. This functional gain plateaus around their physiological values in real islets, at n(beta) approximately 100, n(c) approximately 6 and g(c) approximately 200 pS. In addition, normal beta-cell clusters are robust against significant perturbation to their architecture, including the presence of non-beta cells or dead beta cells. In clusters with n(beta)> approximately 100, coordinated beta-cell bursting can be maintained at up to 70% of beta-cell loss, which is consistent with laboratory and clinical findings of islets. Our results suggest that the bursting characteristics of a beta-cell cluster depend quantitatively on its architecture in a non-linear fashion. These findings are important to understand the islet bursting phenomenon and the regulation of insulin secretion, under both physiological and pathological conditions.     

Targeting prostate cancer based on signal transduction and cell cycle pathways

Prostate cancer remains a leading cause of death in men despite increased capacity to diagnose at earlier stages. After prostate cancer has become hormone independent, which often occurs after hormonal ablation therapies, it is difficult to effectively treat. Prostate cancer may arise from mutations and dysregulation of various genes involved in regulation signal transduction (e.g., PTEN, Akt, etc) and the cell cycle (e.g., p53, p21Cip1, p27Kip1, Rb, etc.). This review focuses on the aberrant interactions of signal transduction and cell cycle genes products and how they can contribute to prostate cancer and alter therapeutic effectiveness.     

Contrasting roles of p57(KIP2) and p21(WAF1/CIP1/SDI1) in transplanted human and bovine adrenocortical cells

Cell transplantation provides a way to compare the regulation of cell proliferation in the same cell type in cell culture and in a vascularized tissue structure in a host animal. The cyclin-dependent kinase inhibitors p57(KIP2), p21(WAF1/CIP1/SDI1) and p27(KIP1) have been extensively studied in cell culture but their role in growth control in tissues is less well understood. In the present experiments we compared the behavior of cell cycle inhibitors in human and bovine adrenocortical cells in culture and following cell transplantation in scid mice. p57 was expressed in the majority of cells in the intact human adrenal cortex. However, double immunofluorescence showed that cells that are in the cell cycle are p57(-) adrenocortical cells, p57 and p27 levels were not affected by inhibition of growth at high cell density, whereas p21 was higher in dividing than growth-inhibited cells. However, p21 was also high in senescent adrenocortical cells. After transplantation of human adrenocortical cells in scid mice, p57 and p27 were observed in most cells in the transplant tissue. Over time the number of p21(+) cells decreased greatly in human adrenocortical cells, but not in bovine adrenocortical cells. This difference correlated with lower levels of cell division (assessed by Ki-67 or incorporation of bromodeoxyuridine) in the human cells in transplant tissues in comparison to bovine cells. The differences between human and bovine cells were observed both when cells were transplanted beneath the kidney capsule and when cells were injected subcutaneously in collagen gel. We conclude that the behavior of p57, but not p21, is consistent with a role as a physiological mediator of proliferative quiescence in the adrenal cortex. The high level of p21 in dividing adrenocortical cells in culture, and in bovine adrenocortical cells in transplant tissues, may be a response to conflicting positive and negative growth influences. Cells may enter the cell cycle under the influence of a strong positive mitogenic signal, but coexisting negative growth stimuli trigger a p21-dependent block to further progression through the cell cycle. This model suggests that bovine adrenocortical cells respond to positive growth stimuli in transplant tissues but human cells lack this response.     

A mathematical model of single target site location by Brownian movement in subcellular compartments

The location of distinct sites is mandatory for many cellular processes. In the subcompartments of the cell nucleus, only very small numbers of diffusing macromolecules and specific target sites of some types may be present. In this case, we are faced with the Brownian movement of individual macromolecules and their "random search" for single/few specific target sites, rather than bulk-averaged diffusion and multiple sites. In this article, I consider the location of a distant central target site, e.g. a globular protein, by individual macromolecules executing unbiased (i.e. drift-free) random walks in a spherical compartment. For this walk-and-capture model, the closed-form analytic solution of the first passage time probability density function (p.d.f.) has been obtained as well as the first and second moment. In the limit of a large ratio of the radii of the spherical diffusion space and central target, well-known relations for the variance and the first two moments for the exponential p.d.f. were found to hold with high accuracy. These calculations reinforce earlier numerical results and Monte Carlo simulations. A major implication derivable from the model is that non-directed random movement is an effective means for locating single sites in submicron-sized compartments, even when the diffusion coefficients are comparatively small and the diffusing species are present in one copy only. These theoretical conclusions are underscored numerically for effective diffusion constants ranging from 0.5 to 10.0 microm(2) s(-1), which have been reported for a couple of nuclear proteins in their physiological environment. Spherical compartments of submicron size are, for example, the Cajal bodies (size: 0.1-1.0 microm), which are present in 1-5 copies in the cell nucleus. Within a small Cajal body of radius 0.1 microm a single diffusing protein molecule (with D=0.5 microm(2) s(-1)) would encounter a medium-sized protein of radius 2.5 nm within 1 s with a probability near certainty (p=0.98).