Detection of apoptotic cells in vitro: a practical standardized experimental protocol using human fibroblasts for initial laboratory studies.

This technical report presents a practical experimental protocol using human fibroblasts that is useful for initiating in situ methods for detection of apoptotic cells in vitro. The protocol details the growth of cells in multichamber wells, and includes a negative control, two positive controls, and untreated fibroblasts to be evaluated for apoptosis localization. Technical tips on handling of solutions, cell culture designs, and separation of slides by treatment are valuable steps for the laboratory beginning such studies. The methods proposed are both time and cost effective.

Anti-apoptotic actions of cytokines in mammalian cells.

Apoptosis is now widely recognized as an important mode of cell death. Since the apoptotic pathway is an active process, modulation of apoptosis is important in our understanding of cell pathophysiology. Recent data have shown the inhibition of apoptosis in different cell types exposed to certain cytokines. Therapeutics that modulate the regulation of apoptosis provide an opportunity for the treatment of certain diseases. There are many reviews for apoptosis induction and the regulators involved. The present report selects important articles on the recent data showing the anti-apoptotic ability of cytokines in mammalian cells. Other novel compounds showing anti-apoptotic functions are also reviewed.

The influence of Matrigel or growth factor reduced Matrigel on human intervertebral disc cell growth and proliferation.

Matrigel (reconstituted basement membrane extract) is a potent inducer of cell growth and differentiation in vitro. This study examined phenotypic variation and proliferative responses of human annular intervertebral disc cells in vitro in Matrigel and Growth Factor Reduced Matrigel (GFR-Matrigel). Cells from age- and gender-matched control subjects and patients with degenerative disc disease were grown either on the surface of, or suspended within, either matrices. Disc cells grew well on top of both matrices with cells spontaneously forming cell projections. Cells grown within either matrix migrated within the gel to form colonies. Increased colony formation within the matrices was seen with young control and patient cells (p < 0.05). Old and young control and patient cells showed increased proliferation within GFR-Matrigel compared to Matrigel. When grown on the matrix surface, young patient and control donor cells showed increased proliferation on GFR-Matrigel compared to Matrigel. Cellular proliferation was significantly greater inside a 3-dimensional environment than a two-dimensional surface monolayer environment. Disc cells had increased proliferation when grown in or on GFR-Matrigel compared to Matrigel. These studies serve as a baseline for subsequent investigations regarding effects of cytokines on disc cells and increase our knowledge of the influence of extracellular matrices on disc cell proliferation.

Effects of pituitary adenylate cyclase-activating polypeptide on hormone secretion by human pituitary adenomas in vitro.

Hormone release in culture in response to pituitary adenylate cyclase-activating polypeptide (PACAP) was examined in 28 human pituitary adenomas: 10 null cell adenomas, 4 gonadotropin-, 6 GH-, 6 ACTH-, and 2 PRL-producing adenomas. The effects of PACAP38 were compared with those of the classical hypothalamic releasing hormones and other activators of intracellular signaling pathways. PACAP38 significantly stimulated GH release from 1 somatotrope tumor (125 +/- 3% of control; P < 0.05) and ACTH release from 3 corticotrope tumors (134 +/- 6%, 136 +/- 7%, and 137 +/- 9% of control; P < 0.05). The effects of PACAP38 were less potent than either GHRH on GH release in the somatotrope tumor or CRH on ACTH release in the corticotrope tumors but similar to the responses seen with the cAMP analog 8-bromo-cAMP (8-Br-cAMP). No detectable effects of PACAP38 on hormone release from null cell, gonadotropin-, or PRL-producing adenomas were observed. Of the 5 somatotrope tumors that failed to respond to PACAP38, all also failed to respond to either 8-Br-cAMP, TRH, or GHRH. Of the corticotrope tumors that failed to respond, 2 of the 3 also failed to respond to CRH. In addition to eliciting hormone release appropriate to the tumor type, PACAP38 also stimulated glycoprotein hormone alpha-subunit (alpha SU) release from one somatotrope tumor (229 +/- 35% of control, P < 0.01) and one corticotrope tumor (149 +/- 4% of control; P < 0.01). This response was not mimicked by 8-Br-cAMP in the somatotrope tumor, but in the corticotrope tumor a significant alpha SU release was also seen after stimulation with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate and 8-Br-cAMP. These results suggest that the novel hypothalamic peptide PACAP38 has a modest role in the regulation of GH, ACTH, and alpha SU secretion from some human tumourous pituitary corticotropes and somatotropes. Further studies are needed to elucidate the intracellular signaling pathways that mediate the effects of PACAP on hormone secretion by these tumor types.

PACAP-38 positively regulates glycoprotein hormone alpha-gene expression in placental cells.

The human glycoprotein hormone alpha-gene is transcriptionally activated by cAMP in placental cells. We have shown that the novel hypothalamic peptide, pituitary adenylate cyclase activating polypeptide, PACAP-38, significantly stimulates intracellular cAMP levels (12-fold increase; P < 0.001) in JEG-3 choriocarcinoma cells. Regulation of alpha-promoter activity was assessed using both the chloramphenicol acetyl transferase (CAT) and the luciferase (LUC) reporter gene systems. alpha-CAT activity was significantly stimulated by PACAP-38 (4-fold increase; P < 0.05) at 24 h with a similar stimulation being seen with a LUC expression vector. The kinetics of stimulation of the alpha-promoter by PACAP-38 were similar to those seen with 8-Br-cAMP and vasoactive intestinal polypeptide (VIP), a peptide which shares 68% homology with PACAP-38. PACAP-38 also stimulated the production of IL-6 from JEG-3 cells with a time course of response similar to that of alpha-promoter transcription. We conclude that human placental choriocarcinoma cells possess functional receptors for PACAP-38, whose activation enhances cAMP formation, alpha-subunit gene transcription and interleukin-6 (IL-6) production.