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Myosin light chain phosphorylation results in cellular contraction and is a critical component of agonist-mediated endothelial cell (EC) junctional gap formation and permeability. We have shown that this reaction is catalyzed by a novel high molecular-weight Ca2+/calmodulin-dependent nonmuscle myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489-494). To characterize EC MLCK expression further in cultured and adult tissues, we employed immunoblotting techniques and reverse transcriptase-polymerase chain reaction to demonstrate that freshly isolated and cultured human macro- and microvascular EC express only the EC MLCK isoform (214 kD), which is distinct from smooth-muscle MLCK isoforms (130 to 150 kD). Immunocytochemical studies demonstrated the presence of the high molecular-weight MLCK isoform in adult human cardiac endothelium using anti-MLCK antibodies, which preferentially recognize the high molecular-weight EC MLCK isoform. Monitoring of MLCK expression in different cell types with antibodies generated against a unique human EC MLCK N-terminal sequence revealed a high level of expression of the 214-kD enzyme in endothelium, minimal level of expression in smooth muscle, and no expression in skeletal muscle. These data suggest that the novel 214-kD kinase, the only MLCK isoform found in endothelium, may be preferentially expressed in this nonmuscle tissue.

We use in situ hybridization to demonstrate that the testicular expression of a novel, mouse, low molecular weight phospholipase A2 (PLA2 Group IIc) mRNA is specific to cells undergoing meiosis. A complete cDNA (1421 bp) encoding the mouse Pla2g2c gene was generated with reverse transcription-PCR (RT-PCR) and 5' and 3' RACE (rapid amplification of cDNA ends) RT-PCR, and its nucleotide sequence was determined. Northern blots of RNA from different tissues revealed a single 1.6 kb transcript only in testis. In situ hybridization indicated that this mouse gene is transcribed mainly in pachytene spermatocytes, secondary spermatocytes, and round spermatids. Expression of the gene is seen in all stages of the seminiferous epithelium, especially in stages VI-VII.

The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

Ovarian cancer (OC) is the most lethal gynecological cancer, characterized by chemo-resistance and fatal tumor recurrence after primary treatment. The metastatic progression is mediated by exfoliation of tumor cells into the peritoneal cavity, the formation of compact OC spheroids, and their implantation onto the overlying peritoneal surface. Spheroid formation is promoted by a change of cell adhesion properties, enhanced secretion of extracellular matrix (ECM) components, such as fibronectin (FN), that stiffen the tumor stroma, promote the aberrant activation of integrins, and recruit the adaptor protein integrin-linked kinase (ILK). ILK drives cytoskeletal assembly and modulates key processes, including survival, invasion, and stemness. However, the functional role of ILK in chemoresistance of ovarian cancer stem cells (OCSCs) remains incompletely understood. We tested the hypothesis that the formation of FN-integrin complexes at the cell membrane promotes survival of OCSCs and supports OC spheroid formation by activating ILK and downstream oncogenic signaling. In OCSCs compared with non-CSC, ILK, FN, and integrin β1 mRNA expression levels were increased (P<0.001) and further enriched in OC spheroids compared with monolayers (P<0.01). ILK blockade with the specific inhibitor cpd-22 suppressed spheroid proliferation and tumor initiating capacity in xenograft mouse model. Furthermore, key oncogenic signaling pathways, in particular decreased β-catenin signaling essential to sustaining cancer cell stemness, were altered in OC spheroids treated with cpd-22. Of the genes examined, expression of Wnt receptor Frizzled 7 (Fzd7) was the mostly highly downregulated in OC spheroids treated with cpd-22, indicating a direct correlation between ILK activation and Wnt signaling. By using co-immunoprecipitation and proximity ligation assay, we demonstrated that ILK co-localizes with Fzd7 in OC spheroids. Mechanistically, treatment of OC spheroids with the combination of carboplatin and ILK-Fzd7 blockade decreased β-catenin signaling, inhibited phospho-Akt at Ser473 and increased levels of cleaved-caspase-3 compared to single agent alone, resulting in apoptosis. By regulating β-catenin signaling, Fzd7 expression and ILK activation are essential for OCSCs survival. Targeting Fzd7-ILK clusters may represent a new therapeutic approach to eradicate OCSC and ultimately improve patient outcomes. Citation Format: Rula Atwani, Virginie Lazar, George Earl Sandusky, Salvatore Condello. Integrin-linked kinase regulates Wnt transcriptional activity in platinum resistant ovarian cancer stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5808.

The mouse autosomal recessive mutant gene weaver (wv) results in abnormalities in cerebellum, substantia nigra and testis. Although a substracted cDNA library prepared by removing P31 (wv/wv) sequences from a P1 (wv/+) library should contain mainly nonrepetitive neonatal sequences, unfortunately, repetitive sequences still appear during screening. Two clones, one repetitive, the other not, are used to illustrate the problems encountered in attempting to isolate the weaver gene from a substrated cDNA library.