Catherine F. Welsh

Timing of cyclin D1 expression within G1 phase is controlled by Rho

The expression of cyclin D1 in mid-G1 phase is associated with sustained ERK activity, and we show here that Rho is required for the sustained ERK signal. However, we also report that Rho inhibits an alternative Rac/Cdc42-dependent pathway, which results in a strikingly early G1-phase expression of cyclin D1. Thus, cyclin D1 is induced in mid-G1 phase because a Rho switch couples its expression to sustained ERK activity rather than Rac and Cdc42. Our results show that Rho is crucial for maintaining the correct timing of cyclin D1 expression in G1 phase and describe a new role for cytoskeletal integrity in the regulation of cell cycle progression.

Rho GTPases as Key Transducers of Proliferative Signals in G1 Cell Cycle Regulation

Mitogenic growth factor- and integrin-dependent signaling pathways cooperate to control the proliferation of nontransformed cells. As integral mediators of these networks, the Rho family of GTPases play a pivotal role in G1 cell cycle progression, primarily through regulation of cyclin D1 expression, as well as the levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1. Such dual control of both the critical positive and negative regulators of G1 progression make the Rho GTPases prime candidates to target the autonomous proliferation which typifies cancer cells. Cyclin D1 has been identified as an important oncogene and the cdk inhibitors as tumor suppressors in human breast carcinogenesis. Evidence pointing to the potential role of Rho-dependent pathways and their interaction with oncogenic Ras in contributing to such cell cycle abnormalities that characterize human breast cancer is also presented.

Relationship between phospholipase C activation and prostaglandin E2 and cyclic adenosine monophosphate production in rabbit tubular epithelial cells. Effects of angiotensin, bradykinin, and arginine vasopressin.

By employing early-passaged rabbit kidney epithelial cells in tissue culture, we demonstrated that angiotensin II (AII) has unique mechanisms of signal transduction. First, unlike its action in other target tissues, micromolar concentrations of AII are required to induce small rises in cytosolic calcium, [Ca2+]i, an action which is not accompanied by the release of inositol phosphates (IP). In contrast, nanomolar bradykinin (BK) mobilizes [Ca2+]i through activation of phospholipase C and release of IP. Neither of these stimulated calcium responses exhibits pertussis toxin (PTx) sensitivity. Secondly, AII and BK at 10(-9) to 10(-7) M stimulate cAMP indirectly through PGE2 production in distal cells. AII- and BK-stimulated PGE2 release is PTx inhibitible, suggestive of the presence of a GTP binding protein mediating the response. By contrast, arginine vasopressin fails to elicit rises in [Ca2+]i but exerts its primary effect on cAMP production in distal cells via direct coupling to a stimulatory GTP binding protein, as evidenced by uncoupling with cholera toxin. Regulation of PGE2 synthesis appears to occur via phospholipase A2, not C, by all three peptides.

Involvement of CD44 and its variant isoforms in membrane-cytoskeleton interaction, cell adhesion and tumor metastasis

SummaryCD44s (standard form of CD44) is a transmembrane glycoprotein whose external domain displays extracellular matrix adhesion properties by binding both hyaluronic acid (HA) and collagen. The cytoplasmic domain of CD44s interacts with the cytoskeleton by binding directly to ankyrin. It has been shown that post-translational modifications, such as phosphorylation (by protein kinase C), acylation (by acyl-transferase) and GTP-binding enhance CD44s interaction with cytoskeletal proteins. Most importantly, the interaction between CD44s and the cytoskeletal protein, ankyrin, is required for the modulation of CD44s cell surface expression and its adhesion function.Recently, a number of tumor cells and tissues have been shown to express CD44 variant (CD44v) isoforms. Using RT-PCR and DNA sequence analyses, we have found that unique CD44 splice variant isoforms are expressed in both prostate and breast cancer cell lines and carcinomas. Most importantly, intracellular ankyrin is preferentially accumulated underneath the patched/capped structures of CD44 variant isoform in both breast and prostate cancer cells attached to HA-coated plates. We propose that selective expression of CD44v isoforms unique for certain metastatic carcinomas and their interaction with the cytoskeleton may play a pivotal role in regulating tumor cell behavior during tumor development and metastasis.

ADP-ribosylation factors: a family of ~20-kDa guanine nucleotide-binding proteins that activate cholera toxin

ADP-ribosylation factors (ARFs) comprise a family of ∼20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of Gsα, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.

A phase II trial of split, low-dose docetaxel and low-dose capecitabine: A tolerable and efficacious regimen in the first-line treatment of patients with HER2/neu-negative metastatic breast cancer

BACKGROUND Successful therapeutic regimens in metastatic breast cancer (MBC) must balance efficacy and tolerability. Docetaxel/capecitabine is an active and commonly used doublet in this setting. Docetaxel upregulates thymidine phosphorylase and thus potentiates the antitumor effects of capecitabine. A schedule with split, low-dose docetaxel in combination with low-dose capecitabine could improve the therapeutic index of this regimen without compromising its clinical activity. PATIENTS AND METHODS Patients with previously untreated HER2/neu-negative MBC were eligible. Treatment consisted of docetaxel 25 mg/m2 on days 1 and 8 in combination with capecitabine 750 mg/m2 twice daily on days 1-14 of a 3-week cycle. Thirty-nine women were enrolled. Median age was 55 years (range, 36-75 years). Fourteen patients had triple-negative disease. Sites of metastasis were as follows: bone (n = 27); liver (n = 15); lung (n = 17); nonregional chest (n = 4); lymph nodes (n = 2); and skin (n = 1). Six patients had bone-only disease. All subjects had a performance status of 0/1. A total of 329 cycles were administered (median, 6; range, 1-50). RESULTS Of 37 patients who received study treatment, 32 had evaluable disease, 1 had a complete response, and 15 had a partial response (overall response rate was 50% in evaluable patients and 43% in the intent-to-treat analysis). Six patients had stable disease. The overall clinical benefit rate was 69% for patients with evaluable disease and 60% overall. Fifteen patients had disease that progressed or had been withdrawn from study at the time of first evaluation. With a median follow-up of 25 months, median time to treatment failure was 4.25 months (95% CI, 1.5-7 months), and median overall survival has not yet been reached. Toxicity was moderate: 15 patients (41%) had grade 3/4 adverse events. CONCLUSION Split, low-dose docetaxel and low-dose capecitabine is an active combination in the first-line treatment of patients with MBC. Toxicity with this schedule was manageable, making it an attractive regimen for further study in combination with targeted agents.

Regulation of G1 to S Phase Transition by Adhesion and Growth Factor Signaling

Progression through the cell cycle is catalyzed by the cyclin-dependent kinases (cdks) whose activities are determined by the relative levels of their cyclin partners and the cyclin-dependent kinase inhibitors. Cyclin D1 paired with cdk4 or 6 and cyclin E paired with cdk2 stimulate progression through G1 (reviewed in Sherr and Roberts, 1999). Cdk inhibitors of the INK4 (p15, p16, p18, and p19) and cip/kip (p21cip l, p27kip1, and p57kip2) families, bind to and inhibit the actions of cyclin D-cdk 4/6 and cyclin E-cdk 2 complexes, respectively. In contrast, cip/kips positively modulate cyclin D-cdk4/ 6 by promoting complex assembly (LaBaer et al., 1997; Cheng et al., 1999). Activated cyclin-cdk complexes sequentially phosphorylate and inactivate the retinoblastoma protein (pRb), a key repressor of G1 phase progression. Cdk-mediated pRb phosphorylation in mid-late G1, phase releases the transcription factor E2F, relieves pRb-mediated repression, and allows for E2F-dependent gene transcription. E2F induces the transcription of a series of genes important for entry into S phase. Phosphorylation of pRb is thought to correspond to passing an operationally defined restriction point, beyond which the cell is irrevocably committed to enter S phase (Pardee, 1989).

Biochemical and Developmental Characterization of ADP-ribosylation Factors, A Family of 20 kDa Guanine Nucleotide-binding Proteins.

The cholera toxin A subunit (CT-A) is an ADP-ribosyltransferase; its principal cellular substrates are the α subunits of Gs, the stimulatory G protein of adenylyl cyclase. The activity of CT-A is enhanced by soluble and membrane proteins termed the ADP-ribosylation factors (ARFs), a family of ~20 kDa guanine nucleotide-binding proteins (Bobak et al., 1990; Kahn and Gilman, 1984; 1986; Tsai et al., 1987; 1988). In the presence of GTP or non-hydrolyzable GTP analogues, but not GDP, its analogues, or adenine nucleotides, bovine brain soluble and membrane ARFs enhanced ADPribosylation of G sα;, simple guanidino compounds, and proteins unrelated to the cyclase system (Tsai et al., 1987; 1988). Kinetic data were consistent with the conclusion that ARF acts as an allosteric activator of the cholera toxin catalytic unit (Noda et al., 1990).