Carol A. Heckman

THE SEVENFOLD WAY OF PKC REGULATION

Protein kinase C (PKC) is a family of enzymes that are physiologically activated by 1,2-diacylglycerol (DAG) and other lipids. To date, 11 different isozymes, alpha, betaI, betaII, gamma, delta, epsilon, nu, lambda(iota), mu, theta and zeta, have been identified. On the basis of their structure and activators, they can be divided into three groups, two of which are activated by DAG or its surrogate, phorbol 12-myristate 13-acetate (PMA). PKC isozymes are remarkably different in number and prevalence in different cell lines and tissues. When activated, the isozymes bind to membrane phospholipids or to receptors that are located in and anchor the enzymes in a subcellular compartment. Some PKCs may also be activated in their soluble form. These enzymes phosphorylate serine and threonine residues on protein substrates, perhaps the best known of which are the myristoylated, alanine-rich C kinase substrate and nuclear lamins A, B and C. The enzymes clearly play a role in signal transduction, and, because of the importance of PMA as a tumor promoter, they are thought to affect some aspect of cell cycling. How PKC takes part in the regulation of cell transformation, growth, differentiation, ruffling, vesicle trafficking and gene expression, however, is largely unknown.

Contact Inhibition Revisited

Contact inhibition of cell movement was originally defined in the 1950s as a way of interpreting studies that were ethological and statistical in nature. Research done in succeeding decades provided a more detailed study of the initial contact and its consequences for the cell. The behavior called contact inhibition is characterized by the cessation of ruffling and forward movement in the lamellipodium of the cell making the contact. A new ruffling membrane then arises elsewhere on the cell perimeter. A comparison between the contact behavior described in the early literature and that of the nerve growth cone, described recently by Steketee and Tosney, suggests that filopodia mediate the sensing function in both cases. Since transformed cells have fewer filopodia than normal cells, the contact behavior may decline in direct response to the degraded function of filopodia. This new “filopodia focal signal transduction” hypothesis of contact inhibition elevates the filopodia sensing function and the cessation of lamellipodial advance to the highest importance as phenomena underlying the altered behavior of cancer cells. J. Cell. Physiol. 220: 574–575, 2009.

Colchitaxel, a coupled compound made from microtubule inhibitors colchicine and paclitaxel

Background Tumor promoters enhance tumor yield in experimental animals without directly affecting the DNA of the cell. Promoters may play a role in the development of cancer, as humans are exposed to them in the environment. In work based on computer-assisted microscopy and sophisticated classification methods, we showed that cells could be classified by reference to a database of known normal and cancerous cell phenotypes. Promoters caused loss of properties specific to normal cells and gain of properties of cancer cells. Other compounds, including colchicine, had a similar effect. Colchicine given together with paclitaxel, however, caused cells to adopt properties of normal cells. This provided a rationale for tests of microtubule inhibitor combinations in cancer patients. The combination of a depolymerizing and a stabilizing agent is a superior anti-tumor treatment. The biological basis of the effect is not understood. Results A single compound containing both colchicine and paclitaxel structures was synthesized. Colchicine is an alkaloid with a trimethoxyphenyl ring (ring A), a ring with an acetamide linkage (ring B), and a tropolone ring (ring C). Although rings A and C are important for tubulin-binding activity, the acetamide linkage on ring B could be replaced by an amide containing a glutamate linker. Alteration of the C-7 site on paclitaxel similarly had little or no inhibitory effect on its biological activity. The linker was attached to this position. The coupled compound, colchitaxel (1), had some of the same effects on microtubules as the combination of starting compounds. It also caused shortening and fragmentation of the + end protein cap. Conclusion Since microtubule inhibitor combinations give results unlike those obtained with either inhibitor alone, it is important to determine how such combinations affect cell shape and growth. Colchitaxel shows a subset of the effects of the inhibitor combination. Thus, it may be able to bind the relevant cellular target of the combination. It will be useful to determine the basis of the shape reversal effect and possibly, the reasons for therapeutic efficacy of microtubule inhibitor combinations.

Persistent effects of phorbol 12‐myristate 13‐acetate: Possible implication of vesicle traffic

Relative to their normal counterparts, transformed epithelial cells have a distinctive and quantifiable three‐dimensional shape. Biophysical and mathematical methods are used to distinguish these extremes in cells from two lines, cultured from rat liver and tracheal epithelium, respectively. Cells adopted a more transformed‐looking configuration transiently when exposed to phorbol 12‐myristate 13‐acetate (PMA) (Plummer and Heckman, [1990] Exp. Cell Res., 188:66–74). The purpose of the present work was to dissect the physiological processes involved in the shape change. Ruffling activity, known to be PMA‐stimulated in other cells, was investigated. Although the ruffles appeared less robust than normal, PMA stimulated ruffling activity over a 5 h period. The number of sites where ruffling was initiated declined by 5 h, however, and suppression was seen by 10 h. Cells from both lines adopted the transformed shape configuration when exposed for 2 h to monensin. When the subset of shape features changed by this treatment was compared with those originally changed during transformation, it was found that monensin‐treated cells mimicked the features of transformed cells. Its effect on ruffling was, however, unlike PMAs. Thus, the phenotype was unlikely to arise from ruffling itself but might be a process driven by ruffling. Chloroquine also stimulated cells to assume characteristics of transformed cells. Since both it and monensin could interfere with endosomes and with the processing of endocytosed contents, this was a likely site of action. Experiments were done to determine whether PMA also affected the processing of extracellular fluids. When the accumulation of horseradish peroxidase (HRP) was measured, the rate was found to be higher in PMA‐treated cells from 5 min, the earliest time assayed, onward. The results suggest that the transformed type of cell in these cell lines showed a constitutive dilation and/or reorganization of some portion of the endosomal pathway.

Relationship of p21-activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21‐activated kinase (PAK)‐dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoAN19 with Cdc42‐associated kinase (ACK). Here, we determine how GTPase‐mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK‐interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK‐dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoAN19 in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK‐mediated autophosphorylation. Coexpression of Nck, ACK, and RhoAN19 abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK–Nck interface. Filopodia were eliminated by the Nck PAK‐binding domain and LPs by the PAK Nck‐binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms. J. Cell. Physiol. 220: 576–585, 2009.

Morphological Fractal Analysis of Shape in Cancer Cells Treated with Combinations of Microtubule-Polymerizing and -Depolymerizing Agents

The current prognostic parameters, including tumor volume, biochemistry, or immunohistochemistry, are not sufficient to reflect the properties of cancer cells that distinguish them from normal cells. Our focus is to evaluate the effects of a combination of microtubule-polymerizing Taxol and -depolymerizing colchicine on IAR20 PC1 liver cells by measuring the surface fractal dimension as a descriptor of two-dimensional vascular geometrical complexity. The fractal dimension offers a rapid means of assessing cell shape. Furthermore, we show correlations of fractal dimensions of cell contours with the latent factors from our previously employed cell shape analysis.

SCANNING ELECTRON MICROSCOPE STUDIES ON FRUITBODY PRIMORDIUM FORMATION IN AGARICUS BISPORUS

To obtain specimens suitable for viewing the early stages of development in Agaricus bisporus, we modified the Halbschalentest method. A plastic foam, impregnated with peat moss, enabled examination ofthe mycelium throughout its entire depth by scanning electron microscopy. Early in the developmental sequence, single hyphae with infrequent branches appeared. As development progressed, the hyphae were consolidated into strands by adhesive, mucilaginous substances. These substances formed electronlucent sheets which were especially obvious after the mycelium was watered. The initial stage of primordium formation was marked by appearance of hyphae which were turgid and tended to curve sharply and branch frequently. These hyphae were found in both initial and later stages of primordium development. Primordia that continued to increase in size appeared to recruit hyphae and strands from surrounding mycelium.

Unraveling the determinants of protrusion formation.

A computerized morphometric classification technique based on latent factors reveals major protrusion classes: factors 4, 5, and 7. Previous work showed that factor 4 represented filopodia, 5 the distribution of lamellar cytoplasm, and 7 a blunt protrusion. We explore the relationship of focal contact (FC) characteristics and their integrated actin cables to factors values. The results show that FC maturation/cytoskeletal integration affects factor 5, because FC elongation/integration was correlated with its values. On the contrary, 7 values decreased with maturation, so cable or FC size or their integration must be restricted to form these protrusions. Where integration did occur, the cables showed distinctive size and orientation, as indicated by correlation of 7 values with FC shape. Results obtained with myosin inhibitors support the interpretation that a central, isometric, contractile network puts constraints on both factor 5 and 7 protrusions. We conclude that cells establish functional domains by rearranging the cytoskeleton.

Effect of Cdc42 domains on filopodia sensing, cell orientation, and haptotaxis

Filopodia are sensors which, along with microtubules, regulate the persistence of locomotion. To determine whether protrusions were involved in sensing adhesion, epithelial cells were cultured on platinum and tantalum gradients. Protrusions were defined by an unbiased statistical method of classification as factors 4 (filopodia), 5 (mass distribution), and 7 (nascent neurites). When the prevalence of protrusions was measured in zones of high (H), middle (M), and low (L) adhesiveness, the main differences were in factor 4. Its values were highest at H and declined at M and L regardless of the gradient composition. The significance of the differences was enhanced when T (top/adhesive end) and B (bottom/nonadhesive end) sides of cells were analyzed separately. Since information about sidedness increased the statistical power of the test, this result suggested that cells pointed more filopodia toward the adhesive end. Trends occurred in factors 5 and 7 only when conditions allowed for a marked trend in factor 4. The data showed that gradient sensing is proportional to the prevalence of filopodia, and filopodia are the only protrusions engaged in comparing adhesiveness across a cell. The probability (P) of the significance of a trend was then used to determine how cells sense the gradient. Binding peptides (BPs) were introduced representing sequences critical for Cdc42 docking on a specific partner. BPs for IQGAP (IQ(calmodulin-binding domain)-containing GTPase-activating protein) and ACK (Cdc42-associated kinase) reduced factor 4 values and prevented cell orientation on the gradient. Micrographs showed attenuated or stubby filopodia. These effectors may be implicated in gradient sensing. Another IQGAP BP increased filopodia prevalence and enhanced orientation on the gradient (P<0.00015). A Wiskott-Aldrich syndrome protein (WASP) BP had no effect. When sensing and orientation were abolished, they both failed at the level of filopodia, indicating that filopodia are both sensors and implementers of signals transduced by adhesion.

Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation

Significance Casitas B-cell lymphoma (Cbl) family proteins are RING finger-containing E3 ubiquitin ligases involved in degradation of activated tyrosine kinases. Previous studies in Cbl-deficient models focused primarily on the consequences of persistent tyrosine kinase signaling resulting in uncontrolled cell activation and proliferation. In the present study, we provide evidence that, in the complete absence of Cbl family proteins, failure to turn over active tyrosine kinases induces irreparable breakdown of the homeostasis of the protein milieu in primary mouse mammary epithelial cells and triggers stress-mediated cell death. Thus, our data reveal that well-regulated removal of active tyrosine kinases is essential for cell survival, an aspect of Cbl family protein functions that has not been previously fully appreciated. Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src.