Loss of PTEN induces microtentacles through PI3K-independent activation of cofilin.

Loss of PTEN tumor suppressor enhances metastatic risk in breast cancer, although the underlying mechanisms are poorly defined. We report that homozygous deletion of PTEN in mammary epithelial cells induces tubulin-based microtentacles (McTNs) that facilitate cell reattachment and homotypic aggregation. Treatment with contractility-modulating drugs showed that McTNs in PTEN(-/-) cells are suppressible by controlling the actin cytoskeleton. Because outward microtubule extension is counteracted by actin cortical contraction, increased activity of actin-severing proteins could release constraints on McTN formation in PTEN(-/-) cells. One such actin-severing protein, cofilin, is activated in detached PTEN(-/-) cells that could weaken the actin cortex to promote McTNs. Expression of wild-type cofilin, an activated mutant (S3A), and an inactive mutant (S3E) demonstrated that altering cofilin phosphorylation directly affects McTNs formation. Chemical inhibition of PI3K did not reduce McTNs or inactivate cofilin in PTEN(-/-) cells. Additionally, knock-in expression of the two most common PI3K-activating mutations observed in human cancer patients did not increase McTNs or activate cofilin. PTEN loss and PI3K activation also caused differential activation of the cofilin regulators, LIM-kinase1 (LIMK) and Slingshot-1L (SSH). Furthermore, McTNs were suppressed and cofilin was inactivated by restoration of PTEN in the PTEN(-/-) cells, indicating that both the elevation of McTNs and the activation of cofilin are specific results arising from PTEN loss. These data identify a novel mechanism by which PTEN loss could remodel the cortical actin network to facilitate McTNs that promote tumor cell reattachment and aggregation. Using isogenic MCF-10A PTEN(-/-) and PIK3CA mutants, we have further demonstrated that there are clear differences in activation of cofilin, LIMK and SSH between PTEN loss and PI3K activation, providing a new evidence that these mutations yield distinct cytoskeletal phenotypes, which could have an impact on tumor biology.

c-Src differentially regulates the functions of microtentacles and invadopodia.

During metastasis, invading cells produce various actin-based membrane protrusions that promote directional migration and proteolysis of extracellular matrix (ECM). Observations of actin staining within thin, tubulin-based microtentacle (McTN) protrusions in suspended MDA-MB-231 tumor cells, prompted an investigation of whether McTNs are structural or functional analogs of invadopodia. We show here that MDA-MB-231 cells are capable of producing invadopodia and McTNs, both of which contain F-actin. Invadopodium formation was enhanced by the expression of a constitutively active c-Src kinase, and repressed by the expression of dominant-negative, catalytically inactive form of c-Src. In contrast, expression of inactive c-Src significantly increased McTN formation. Direct inhibition of c-Src with the SU6656 inhibitor compound also significantly enhanced McTN formation, but suppressed invadopodia, including the appearance of F-actin cores and phospho-cortactin foci, as well as completely blocking focal degradation of ECM. In addition, silencing of Tks5 in Src-transformed fibroblasts blocked invadopodia without affecting McTNs. Genetic modification of c-Src activity that promoted McTN formation augmented capillary retention of circulating tumor cells in vivo and rapid re-attachment of suspended cells in vitro, even though invadopodia were strongly suppressed. These results indicate that McTNs are capable of enhancing tumor cell reattachment, even in the absence of Tks5 and active Src, and define separate cytoskeletal mechanisms and functions for McTNs and invadopodia.

Antibacterial drug discovery: is small pharma the solution?

Although antibacterial research has declined in many larger pharmaceutical companies, small companies have begun to fill in the gap. Antibacterial discovery research is currently being conducted in at least 35 small companies. One successful approach taken by small pharma has been to continue clinical programmes that were abandoned by large companies. Issues surrounding these activities, as well as proposed changes, are outlined.

Discovery of MC-02,331, a new cephalosporin exhibiting potent activity against methicillin-resistant Staphylococcus aureus.

A systematic approach toward building activity against methicillin-resistant staphylococci into the cephalosporin class of beta-lactam antibiotics is described. Initial work focused on finding the optimal linkage between the cephem nucleus and a biphenyl pharmacophore, which established that a thio linkage afforded potent activity in vitro. Efforts to optimize this activity by altering substitution on the pharmacophore afforded iodophenylthio analog MC-02,002, which although highly potent against MRSA, was also highly bound to serum proteins. Further work to decrease serum protein binding showed that replacement of the iodo substituent by the positively-charged isothiouronium group afforded potent activity and reduced serum binding, but insufficient aqueous solubility. Solubility was enhanced by incorporation of a second positively-charged group into the 7-acyl substituent. Such derivatives (MC-02,171 and MC-02,306) lacked sufficient stability to staphylococcal beta-lactamase enzymes. The second positive charge was incorporated into the cephem 3-substituent in order to utilize the beta-lactamase-stable aminothiazolyl(oximino)acetyl class of 7-substituents. These efforts culminated with the discovery of bis(isothiouroniummethyl)phenylthio analog MC-02,331, whose profile is acceptable with respect to potency against MRSA, serum binding, aqueous solubility, and beta-lactamase stability.

Recall intervals: effect on treatment needs: a retrospective study.

The purpose of this retrospective study was to examine the effect of recall intervals on the incidence of dental caries. Data were collected from patient records of a private pediatric dental practice. Variables examined were time of the recall interval, age, race and sex of the patient, and whether the patient lived in a fluoridated area. There were 207 patients who qualified for the study. Of the 207 patients in the study, 173 did not have any teeth with dental caries at the recall visit. A significant difference between increased caries activity and recall interval was not found in this study. There was no significant difference found between the explanatory variables and caries activity.

Recall intervals: effect on treatment needs of the handicapped patient: a retrospective study.

The purpose of the retrospective study was to examine the effect of recall intervals on incidence of dental caries in handicapped patients. Data was collected from patient records of a private pediatric dental practice. Variables examined were time of the recall interval, age, race and sex of the patient, handicap, and whether the patient lived in a fluoridated area or not. Approximately six hundred charts were reviewed which resulted in 83 patients that qualified for the study. Of the 83 patients in the study, 57 did not have dental caries at the recall visit. The relationship between increased caries activity and recall interval was not significant. However, a trend indicating an increased chance of developing caries after a twelve month recall interval was detected.

Vacuolated anterior horn cells in wobbler mouse motor neuron disease: peripheral axons and regenerative capacity.

We investigated whether vacuolated cervical anterior horn cells of the wobbler mouse maintain axons to the periphery, and if these morphologically abnormal neurons are capable of supporting axonal regeneration. Using retrograde axonal transport, we applied horseradish peroxidase (HRP) to peripheral nerves or muscles and with electron microscopy sought evidence for perikaryal labeling in vacuolated neurons in 23 wobbler mice. When HRP was injected into forelimb muscles, 12 of 36 vacuolated neurons became positively labeled indicating that these neurons have axons in continuity with the periphery. In regeneration studies, after nerve crush at the brachial plexus, 23 out of 85 vacuolated neurons were labeled after HRP application at the elbow level. However, after a sufficient regeneration period, none of the 36 vacuolated neurons were labeled if HRP was applied in muscles below the elbow. In all experiments, morphologically normal neurons were always labeled. Our studies indicate that some vacuolated neurons of wobbler mice not only maintain axons into the periphery, but are also capable of supporting regeneration. However, the overall function of these vacuolated neurons appears marginal compared with the majority of morphologically normal neurons in this motor neuron disease.