Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW-and LMW-HA, respectively). These different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW-and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.

Experiencing some early life adversity can have an “inoculating” effect that promotes resilience in adulthood. However, the mechanisms underlying stress inoculation are unknown, and animal models are lacking. Here we used the limited bedding and nesting (LBN) model of adversity to evaluate stress inoculation of addiction-related phenotypes. In LBN, pups from postnatal days 2 to 9 and their dams were exposed to a low-resource environment. In adulthood, they were tested for addiction-like phenotypes and compared to rats raised in standard housing conditions. High levels of impulsivity are associated with substance abuse, but in males, LBN reduced impulsive choice compared to controls. LBN males also self-administered less morphine and had a lower breakpoint on a progressive ratio reinforcement schedule than controls. These effects of LBN on addiction-related behaviors were not found in females. Because the nucleus accumbens (NAc) mediates these behaviors, we tested whether LBN altered NAc physiology in drug-naïve and morphine-exposed rats. LBN reduced the frequency of spontaneous excitatory postsynaptic currents in males, but a similar effect was not observed in females. Only in males did LBN prevent a morphine-induced increase in the AMPA/NMDA ratio. RNA sequencing was performed to delineate the molecular signature in the NAc associated with LBN-derived phenotypes. LBN produced sex-specific changes in transcription, including in genes related to glutamate transmission. Collectively, these studies reveal that LBN causes a male-specific stress inoculation effect against addiction-related phenotypes. Identifying factors that promote resilience to addiction may reveal novel treatment options for patients.

Human breast tumors often exist in an acidic and hypoxic microenvironment, which can promote resistance to radiation and chemotherapies. A tumor-selective pH gradient arises in these tumors which favors uptake and retention of drugs like camptothecin that are weak acids. We evaluated the effect of alkyl substitutions at the 7 position in seven CPTs with varying groups at the 10 position on modulation by acidic extracellular pH in three human breast cancer cell lines. Growth inhibition was assessed by propidium iodide staining of nucleic acids in human breast cancer cells cultured at either extracellular pH 6.8 or 7.4 that were (1) hormone-sensitive (MCF-7/wt), (2) hormone insensitive (MDA-MB-231), or (3) alkylator-resistant (MCF-7/4-hc). Over 10-fold pH modulation was observed in 7-halomethyl analogs of methylenedioxy-CPT and in 7-alkyl analogs of 10-amino-CPT. Of 39 analogs tested, the overall pattern of activity across breast tumor cell lines was similar with some notable exceptions. For example, 7-propyl-10-amino-CPT was modulated 16- to 20-fold by acidic extracellular pH in the MCF-7 cell lines, but only 6-fold in MDA-MB-231 cells. One mechanism that can contribute to pH modulation is enhanced cellular drug uptake and retention. In MCF-7/wt cells, uptake of 10-amino-CPT increased 4-fold, while retention increased over 10-fold at acidic extracellular pH. In addition, gene expression analysis of MCF-7/wt cells indicated that expression of a number of genes changed under acidic culture conditions, including down-regulation of the CPT efflux protein pump breast cancer resistance protein (BCRP). Interestingly, expression of topoisomerase I, the molecular target of CPT, was not affected by acidic growth conditions. These results highlight the importance of maintaining key features of tumor physiology in cell culture models used to study cancer biology and to discover and develop new anticancer drugs. While several substitutions at the 7 and 10 positions enhance potency, 7-halomethyl and 10-amino CPT analogs show selective activity at the acidic pH common to the microenvironment of most solid tumors.

Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity. The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro. Furthermore, these findings suggest that chloroethylaziridine may also contribute to the toxicity of cyclophosphamide in vivo.

The DNA antimetabolite gemcitabine is an anticancer agent with shown preclinical and clinical utility and a low toxicity profile. In this study, we sought to identify and optimize drug partners for binary and tertiary combinations with gemcitabine for use in the treatment of acute myelogenous leukemia (AML). Drug interaction was assessed by growth inhibition assay with metabolic end points. The combination index method was used to evaluate combinations of gemcitabine with fludarabine, paclitaxel, chlorambucil, doxorubicin, mitoxantrone, and SN-38 in U937 human AML cells. A three-dimensional method was used to determine the effect of dose ratio and schedule on drug interaction. Mechanisms underlying interactions related to cell cycle effects and apoptosis were assessed by flow cytometric and caspase-3 and -7 assays, respectively. The most synergistic binary combination was gemcitabine + fludarabine.The most synergistic tertiary combination was gemcitabine + fludarabine + paclitaxel, where the interaction was sequence dependent with paclitaxel given before gemcitabine + fludarabine, producing a 2-fold increase in synergy. Cell cycle analysis did not reveal a significant G 2 -M arrest, suggesting that the synergistic effect of paclitaxel in this combination, which produced the greatest caspase activation, might be independent of microtubule stabilization. In contrast, the gemcitabine + fludarabine + mitoxantrone combination was synergistic and schedule independent. Moreover, few ratios of gemcitabine + fludarabine to mitoxantrone were antagonistic, which could be important for clinical translation. In conclusion, synergistic interactions with gemcitabine occurred with several drugs, the most promising being gemcitabine + fludarabine, gemcitabine + fludarabine + paclitaxel, and gemcitabine + fludarabine + mitoxantrone. These findings provided a rationale for clinical trials of gemcitabine + fludarabine and gemcitabine + mitoxantrone where responses were observed in heavily pretreated AML patients.Leukemia is one of the 10 leading causes of cancer deaths in the United States, and acute myelogenous leukemia (AML) is responsible for one third of these deaths (1). Whereas several promising compounds have recently become available, singleagent therapies have been largely unsuccessful in preventing relapse (2). When optimized, combination chemotherapy regimens can offer increased efficacy, decreased toxicity, dose reductions, and decreased drug resistance.Standard induction chemotherapy consists of cytosine arabinoside in combination with an anthracycline antibiotic or anthracenedione (usually daunorubicin, idarubicin, or mitoxantrone). Gemcitabine (2V ,2V -difluorodeoxycytidine) is structurally similar to cytosine arabinoside, but creates DNA damage that is more difficult to repair. In addition, gemcitabine metabolites inhibit ribonucleotide reductase, leading to selfpotentiation of cytotoxic activity (3). Gemcitabine has proven successful against a wide range of tumor types including lung, ovarian, head and nec...