Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone.

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin I beta) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

Combined depletion of cell cycle and transcriptional cyclin-dependent kinase activities induces apoptosis in cancer cells.

Selective cyclin-dependent kinase (cdk) 2 inhibition is readily compensated. However, reduced cdk2 activity may have antiproliferative effects in concert with other family members. Here, inducible RNA interference was used to codeplete cdk2 and cdk1 from NCI-H1299 non-small cell lung cancer and U2OS osteosarcoma cells, and effects were compared with those mediated by depletion of either cdk alone. Depletion of cdk2 slowed G1 progression of NCI-H1299 cells and depletion of cdk1 slowed G2-M progression in both cell lines, with associated endoreduplication in U2OS cells. However, compared with the incomplete cell cycle blocks produced by individual depletion, combined depletion had substantial consequences, with G2-M arrest predominating in NCI-H1299 cells and apoptosis the primary outcome in U2OS cells. In U2OS cells, combined depletion affected RNA polymerase II expression and phosphorylation, causing decreased expression of the antiapoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis (XIAP), effects usually mediated by inhibition of the transcriptional cdk9. These events do not occur after individual depletion of cdk2 and cdk1, suggesting that reduction of cdk2, cdk1, and RNA polymerase II activities all contribute to apoptosis in U2OS cells. The limited cell death induced by combined depletion in NCI-H1299 cells was significantly increased by codepletion of cdk9 or XIAP or by simultaneous treatment with the cdk9 inhibitor flavopiridol. These results show the potency of concomitant compromise of cell cycle and transcriptional cdk activities and may guide the selection of clinical drug candidates.

Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions.

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.

Flavopiridol-induced apoptosis during S phase requires E2F-1 and inhibition of cyclin A-dependent kinase activity.

Transformed cells are selectively sensitized to apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol after their recruitment to S phase. During S phase, cyclin A-dependent kinase activity neutralizes E2F-1 allowing orderly S phase progression. Inhibition of cyclin A-dependent kinase by flavopiridol could cause inappropriately persistent E2F-1 activity during S phase traversal and exit. Transformed cells, with high baseline levels of E2F-1 activity, may be particularly sensitive to cyclin A-dependent kinase inhibition, as the residual level of E2F-1 activity that persists may be sufficient to induce apoptosis. Here, we demonstrate that flavopiridol treatment during S phase traversal results in persistent expression of E2F-1. The phosphorylation of E2F-1 is markedly diminished, whereas that of the retinoblastoma protein is minimally affected, so that E2F-1/DP-1 heterodimers remain bound to DNA. In addition, manipulation of E2F-1 levels leads to predictable outcomes when cells are exposed to flavopiridol during S phase. Tumor cells expressing high levels of ectopic E2F-1 are more sensitive to flavopiridol-induced apoptosis during S phase compared with parental counterparts, and high levels of ectopic E2F-1 expression are sufficient to sensitize nontransformed cells to flavopiridol. Furthermore, E2F-1 activity is required for flavopiridol-induced apoptosis during S phase, which is severely compromised in cells homozygous for a nonfunctional E2F-1 allele. Finally, the response to flavopiridol during S phase is blunted in cells expressing a nonphosphorylatable E2F-1 mutant incapable of binding cyclin A, suggesting that the modulation of E2F-1 activity produced by flavopiridol-mediated cyclin-dependent kinase inhibition is critical for the apoptotic response of S phase cells.

A phase II clinical and pharmacodynamic study of E7070 in patients with metastatic, recurrent, or refractory squamous cell carcinoma of the head and neck: modulation of retinoblastoma protein phosphorylation by a novel chloroindolyl sulfonamide cell cycle inhibitor.

PURPOSE: E7070 is a synthetic sulfonamide cell cycle inhibitor that induces hypophosphorylation of the retinoblastoma (Rb) protein and G(1) arrest in vitro. This Phase II study was conducted to explore the efficacy, safety, and pharmacodynamics of E7070 in squamous cell carcinoma of the head and neck (SCCHN). EXPERIMENTAL DESIGN: Patients with metastatic, recurrent, or refractory SCCHN, treated with no more than one prior therapy for recurrent disease, received E7070 at 700 mg/m(2) over 1 h every 3 weeks. Pre- and posttreatment tumor fine needle aspirates were subjected to immunohistochemistry with a panel of phospho-specific anti-Rb antibodies. End points included progression-free survival, response rate and duration, overall survival, toxicity profile, and inhibition of Rb phosphorylation. RESULTS: Because none of the first 15 patients achieved progression-free survival > 4 months, the early stopping rule was invoked. Eleven patients had oropharyngeal cancer and 12 were male. Median age was 59 years (range, 49-73 years). Thirty-nine cycles of E7070 were delivered (median, 2.6 cycles/patient; range, 1-5 cycles). Six patients had stable disease after 2 cycles and 2 patients each subsequently received 1, 2, and 3 additional cycles, respectively, before experiencing progression. Immunohistochemistry of tumor cell aspirates from 3 patients demonstrated reduced Rb phosphorylation posttreatment. CONCLUSIONS: At this dose and schedule, E7070 is unlikely to be superior over single-agent chemotherapy in SCCHN. However, the data suggest that cdk activity can be inhibited in tumor cells, resulting in posttreatment modulation of Rb phosphorylation. In the absence of cytotoxicity, more frequent administration of E7070 may be required to sustain Rb hypophosphorylation and cytostatic growth arrest.