Lee E. Babiss

[81] Measurement of the effect of interferons on cellular differentiation of human skeletal muscle cells

Publisher Summary This chapter discusses the measurement of the effect of interferons on cellular differentiation of human skeletal muscle cells. Improved cell culture techniques permit the routine cultivation of human skeletal muscle cells in vitro . During ontogeny and regeneration of skeletal muscle in vivo , myogenic mononuclear cells fuse spontaneously to form multinucleated syncytia (myotubes) that no longer undergo conventional DNA synthesis. As myoblasts fuse into myotubes, several muscle-specific proteins are produced while the synthesis of nonmuscle proteins is reduced. A similar process also occurs in cultures prepared from both fetal and neonatal tissue. It is even possible to establish differentiating muscle cultures from mature skeletal muscle, because dormant mononuclear myogenic cells (called “muscle-satellite cells”), that are present with the muscle fibers, remain present throughout life. Although several muscle-specific markers can be analyzed for evaluating myogenesis in culture, Creatine Kinase (CK) isozyme analysis is among the most convenient, because isozyme transitions begin to appear rather early in the differentiation process, and the fluorescent isozyme assay is sensitive, rapid, and rather inexpensive. CK isozyme test kits, produced for serum assays in the clinical laboratory by several companies, can be used satisfactorily for tissue culture homogenates, thereby, eliminating the time-consuming process of preparing reagents.

[80] Measurement of the effect of interferons on cellular differentiation in murine and human melanoma cells

Publisher Summary This chapter describes some techniques for evaluating the effect of interferon or other compounds on melanogenesis in murine and human melanoma cell cultures. Mouse and human systems should prove useful in investigating the effect of modulators of differentiation, such as phorbol esters, mezerein (MEZ), and interferon, on growth, gene expression, and differentiation as well as serve as models for studying potential interrelationships between these processes. With the B-16 and human melanoma cell culture systems, it is possible to study the effect of virtually any compound or combination of compounds, which is soluble in tissue culture medium on the process of melanogenesis. These model systems are valuable in defining the role of extrinsic signals in modulating differentiation, in examining at the genetic level (i.e., transcription and processing of messenger RNA (mRNA)) the role of specific genes in the differentiation process, and in examining the role of transmembrane signaling in regulating differentiation. Cell culture systems are currently available for analyzing the molecular and biochemical events associated with the specific programs of cell differentiation. With these systems, it should now be possible to determine the role of endogenous compounds, such as hormones and interferon, as well as the effect of various exogenously applied agents, such as chemotherapeutic drugs, growth factors and viruses, on differentiation.

Suppression of the progression phenotype in somatic cell hybrids occurs in the absence of altered adenovirus type 5 gene expression

In the present study we have analyzed the genetic regulation of increased expression of transformation-associated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (E11-NMTneo) and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFcap). Parental E11-NMTneo cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFcap cells exhibited phenotypes opposite to those of E11-NMTneo cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFcap and E11-NMTneo cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which still exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with E11-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 E1A and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in Ad5-transformed cells and that this suppression is not associated with changes in the expression of Ad5 transforming genes.

Abstract LB-234: Suppression of Bruton tyrosine kinase with X-022, a highly selective next-generation irreversible inhibitor

BTK is a member of the TEC family of kinases and is a critical component of B cell receptor (BCR) signaling, promoting B cell maturation, survival and proliferation. BCR signaling is requisite in all B cell-mediated malignancies where clinical trials with BTK inhibitors have proven efficacious. X-Rx utilized its drug discovery platform and knowhow to advance multiple novel series of BTK inhibitors. X-022 was then identified as a next generation, oral small molecule BTK inhibitor. X-022 irreversibly inhibits BTK with an IC50 of 2.3 nM. In primary human B cell and HWB assays, X-022 inhibited B cell activity with IC50 values of 49 and 93 nM, respectively. X-022 showed superior selectivity against a panel of 456 kinases, only inhibiting at >90% BTK at a concentration of 1000 nM. Oral bioavailability of >20% is observed in mice, rats and dogs. In a mouse PK/PD model, X-022 showed a dose dependent decrease in B cell activation measured over an 8 hour time interval with >98% inhibition observed at 10 mg/kg po. In an established model of collagen induced arthritis in mice, X-022 caused a dose dependent decrease in disease severity consistent with mechanism of action. No safety issues were identified in a pilot study in rodents at doses up to 300 mg/kg po. X-022 is a promising next generation irreversible oral BTK inhibitor for the treatment of B cell malignancies and chronic autoimmune diseases. In addition, the high degree of selectivity combined with favorable molecular properties make X-022 an excellent candidate for co-formulation with other targeted therapeutics. Citation Format: Mark J. Mulvihill, Xiangyang Chen, JP Shaw, Richard Liu, Haihong Ni, Lee Babiss, Louis Renzetti. Suppression of Bruton tyrosine kinase with X-022, a highly selective next-generation irreversible inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-234. doi:10.1158/1538-7445.AM2014-LB-234