Opioid growth factor regulates the cell cycle of human neoplasias.

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

Angiotensinogen and AT(1) antisense inhibition of osteopontin translation in rat proximal tubular cells.

Antisense oligonucleotide inhibition of angiotensinogen and ANG II type 1 receptor (AT(1)) mRNA translation in rat proximal tubules (PT) was examined to provide direct evidence for a role of the renin-angiotensin system (RAS) in upregulated osteopontin expression observed following mechanical cell stretch. Male Sprague-Dawley rats underwent unilateral ureteral obstruction (UUO) under Brevital anesthesia. In situ hybridization and Western blot analysis demonstrated angiotensinogen mRNA and angiotensin converting enzyme (ACE) protein localized to PTs and upregulated in obstructed kidneys, respectively, confirming an increased expression of renal RAS in vivo. In vitro studies were performed to provide mechanistic insight into ANG II-dependent osteopontin expression following mechanical cell stretch, which putatively mimics the increased PT luminal pressure post-UUO. A cationic transfection method was used to introduce either angiotensinogen or AT(1) antisense oligonucleotide into cultured rat PT cells prior to 1 h of cyclic mechanical cell stretch. Northern blot analysis revealed that PT cells subjected to cyclic mechanical stretch with/without prior transfection with a sense oligonucleotide exhibited increased osteopontin mRNA expression compared with unstretched cells. Blockade of either angiotensinogen or AT(1) mRNA translation by antisense oligonucleotide inhibition prior to cell stretch was found to significantly decrease osteopontin mRNA levels 2.4-fold (P<0.004) and 1.6-fold (P<0.001), respectively, compared with values observed in control unstretched cells. This study provides evidence that stretch-induced upregulation of osteopontin mRNA expression is mediated, in part, via production of ANG II. These results lend insight into upregulation of osteopontin via a local PT RAS leading to macrophage infiltration in the tubulointerstitium in experimental hydronephrosis.

Confocal microscopic observation of cytoskeletal reorganizations in cultured shark rectal gland cells following treatment with hypotonic shock and high external K+.

The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has served as a model experimental system for investigating the relationship between the actin cytoskeleton and cell volume regulation. Previous reports employing conventional fluorescence microscopy of tissue slices have shown that cells exposed to high external K+ and hypotonically-induced cell swelling displayed a fading of F-actin staining intensity, particularly at the basolateral cell borders. However, spectroscopic measurement of the F-actin present in similarly treated rectal gland slices failed to demonstrate a net change in F-actin amount. In an effort to resolve the structural reorganizations of F-actin which may be occurring during high K+ and hypotonic shock treatments, we have used cultured SRG cells in conjunction with confocal microscopic immunocytochemical localization techniques to examine actin filament, microtubule, and cytokeratin filament dynamics under these two experimental conditions. The results reveal that F-actin in control cells exists in an array of parallel linear bundles (which do not appear to be stress fiber-like given their lack of staining for myosin II or alpha-actinin) that is reorganized to a punctate pattern in hypotonic shock and a dense meshwork in high K+. The linear bundle pattern of F-actin returns in cells undergoing regulatory volume decrease. Quantitative western blotting of F-actin in SRG cell detergent extracted cytoskeletons indicates no significant difference in the relative amounts of F-actin present in control, hypotonic shocked, or high K+ cells. Anti-tubulin and anti-cytokeratin labeling of the treated SRG cells suggest that these other major cytoskeletal elements are not significantly altered by the treatments. Taken together, our results reinforce the concept that there is an association between the structural organization of the actin cytoskeleton and cell volume regulation in the SRG epithelial cells.

The heterotrimeric motor protein kinesin-II localizes to the midpiece and flagellum of sea urchin and sand dollar sperm.

We have utilized immunoblotting and light microscopic immunofluorescent staining methods to examine the expression and localization of sea urchin kinesin-II, a heterotrimeric plus end-directed microtubule motor protein (previously referred to as KRP(85/95)), in sea urchin and sand dollar sperm. We demonstrate the presence of the 85 K and 115 K subunits of kinesin-II in sperm and localize these proteins to the sperm flagella and midpiece. The kinesin-II localization pattern is punctate and discontinuous, and in the flagella it is quite distinct from the continuous labeling present in sperm labeled with anti-flagellar dynein. The kinesin-II staining is largely insensitive to prefixation detergent extraction, suggesting that it is not associated with membranous elements in the sperm. In the midpiece the kinesin-II staining is similar to the pattern present in sperm labeled with an anti-centrosomal antibody. To our knowledge, this is the first localization of kinesin-like proteins in mature sperm and corroborates the recent identification and localization of kinesin-like proteins in the flagella and basal body of the unicellular green alga Chlamydomonas. We hypothesize that kinesin-II in the sperm may play functional roles in intraflagellar transport and/or the formation of flagella during spermatogenesis.