Barry L. Brown

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.

Factors affecting the saturation assay of cyclic AMP in biological systems

Abstract Nonspecific (blank) effects interfering with the saturation analysis of cyclic AMP may be mediated either through the primary reaction between cyclic AMP and the binding protein or on the efficacy of the separation procedure. It is not normally permissible to substract blank values, measured at zero concentration of added nucleotide from the measurement of unknowns since the magnitude of the correction may vary with different nucleotide concentrations. Response curves should be set up in equivalent concentrations of the particular blank extract in every assay.

Modulation of cyclic AMP accumulation in GH3 cells by a phorbol ester and thyroliberin

4 beta phorbol-12, 13-dibutyrate (PDBu) stimulated cyclic AMP accumulation in GH3 pituitary tumour cells in the presence of isobutylmethylxanthine. This effect persisted after preincubation of cells with cholera or pertussis toxins. In contrast, vasoactive intestinal polypeptide (VIP)-stimulated cyclic AMP accumulation was inhibited by PDBu in a dose dependent fashion (IC50 = 5.1 nM). Thyroliberin (TRH) had a similar, but non-additive, stimulatory effect on cyclic AMP accumulation with PDBu, however it did not inhibit VIP stimulation. These results suggest that TRH may stimulate cyclic AMP accumulation through protein kinase C and that stimulation of adenylate cyclase by PDBu and TRH may occur distal to the guanine nucleotide binding regulatory proteins, Ns and Ni.

The survival effect of prolactin on PC3 prostate cancer cells.

OBJECTIVES Recent studies suggest a paracrine/autocrine loop involving prolactin (PRL) within the human prostate. The aims of this study were to determine the effects of PRL on the growth and survival of prostate cancer cells and the intracellular signalling mechanisms underlying such effects. METHODS The effect of PRL on proliferation of LNCaP, PC3 and DU145 was assessed by Coulter counting. The effect of PRL on TRAIL-, staurosporine- and flavopiridol-induced apoptosis was assessed by Timelapse microscopy and Annexin V binding. The status of the PRL receptor (PRL-R) and Akt/PKB (protein kinase B) activity were assessed by Western blotting. RESULTS All three cell lines expressed both the short and long forms of the PRL receptor. Although, no significant effect of PRL on the proliferation of these cells was found, PRL partially inhibited TRAIL-induced apoptosis in PC3 cells. PRL also enhanced the phosphorylation of Akt/PKB in these cells. CONCLUSIONS PRL had no significant effect on the proliferation of PC3, DU145 and LNCaP, but inhibited TRAIL-induced apoptosis in PC3 cells, possibly via enhanced Akt/PKB phosphorylation in PC3 cells. Further investigations are underway to determine the survival effect of PRL on the other two prostate cancer cell line.

Bradykinin stimulates the production of prostaglandin E2 and interleukin-6 in human osteoblast-like cells

The effect of bradykinin (BK) on proteinase activity, prostaglandin synthesis, and the production of interleukin-6 (IL-6) was investigated in cultures of human osteoblast-like cells. Bradykinin had no effect on stromelysin activity and plasminogen activator activity produced by human osteoblast-like cells. However, BK stimulated the production of prostaglandin E2, an effect that was markedly enhanced by pre-incubation with recombinant interleukin-1 alpha (rhIL-1 alpha), but was apparently unaffected by BK receptor antagonists types 1 and 2. Bradykinin stimulated the intracellular accumulation of total inositol phosphates suggesting that its effects were mediated by stimulation of phosphoinositide metabolism. Bradykinin within the dose range of 10(-11)-10(-5) M also significantly stimulated the production of IL-6. Bradykinin may, therefore, mediate a variety of responses in bone under both physiological and pathological conditions.

Prognostic significance of Akt, phospho-Akt and BAD expression in primary breast cancer

Apoptotic markers in breast cancer are reported to have prognostic significance. The aim of this study was to assess the prognostic value of Akt, phospho-Akt and BAD expression in primary tumours from breast cancer patients. Expression of phospho-Akt did not correlate with menopausal status, nodal involvement or tumour size, although there was a significant correlation between phospho-Akt and oestrogen receptor status and tumour grade. No association was found between phospho-Akt and BAD. However, a significant correlation was found between Akt and BAD. Akt and phospho-Akt expression did not correlate with either disease-free survival (DFS) or overall survival (OS). Conversely, BAD immunostaining correlated significantly with increasing tumour size and with oestrogen receptor (ER) immunostaining in both frozen and paraffin sections. Expression of BAD appeared to be nucleolar in addition to its cytoplasmic and nuclear staining. Comparison of immunohistochemical staining on frozen sections and paraffin sections showed a reasonable concordance in Akt and BAD immunoreactivity. However, the results showed for the first time that strong BAD expression is related to a favourable prognosis but is not an independent prognostic factor. In conclusion, these results could provide the basis for understanding how Akt, phospho-Akt and BAD expression contributes to the prognosis of invasive breast cancer.

Enhanced anti-cancer effect of a phosphatidylinositol-3 kinase inhibitor and doxorubicin on human breast epithelial cell lines with different p53 and oestrogen receptor status.

New efforts are being focused on signalling pathways as targets for cancer therapy. This particular study was designed to investigate whether blockade of the phosphatidylinositol 3OH‐kinase (PI3K) pathway (a survival/anti‐apoptosis pathway, overexpressed in various tumours) could sensitise human breast cancer cells to the effect of chemotherapeutics. Doxorubicin (Dox) and LY294002 (LY, a PI3K inhibitor) were used individually or in combination on MDA‐MB‐231 (p53 mutant, ER‐), T47D (p53 mutant, ER+), and MCF‐7 (p53 wildtype, ER+) human breast cancer cell lines, and on 184A1, a nonmalignant human breast epithelial cell line (p53 wildtype, ER‐). Each drug showed time‐ and dose‐dependent growth inhibition of cell proliferation on all 4 cell lines. The combination of Dox+LY resulted in enhanced cell growth inhibition in MDA‐MB‐231 and T47D cells, and additive inhibition in MCF‐7 and 184A1 cells. Cell cycle analysis showed that Dox+LY enhanced the arrest of MDA‐MB‐231 and T47D cells in G2 with the appearance of a sub‐G1 peak indicating apoptosis/necrosis, a notion supported by enhanced depolarisation of mitochondrial membrane potential in these cell types. The combination also caused a greater additive increase in Cyclin B1. Thus, the synergistic effect of the combination on cell proliferation in some, but not all, breast cancer cells may be through enhanced induction of both G2 arrest and apoptosis, in which p53 may play a role. Substantially lower doses of doxorubicin could be used with low doses of inhibitors of the PI3K pathway, without compromising the anti‐cancer effect, but also lowering detrimental side‐effects of doxorubicin. This study supports the notion that survival signalling pathways offer special targets for chemotherapy in cancer.

Muscarinic acetylcholine receptor activation causes inhibition of cyclic AMP accumulation, prolactin and growth hormone secretion in GH3 rat anterior pituitary tumour cells.

Acetylcholine, oxotremorine and carbachol, compounds that exhibit muscarinic agonist activity, maximally inhibited basal prolactin secretion from GH3 cells by approx. 50% and intracellular cyclic AMP levels by approx. 20%. Both parameters were inhibited with similar potencies by each agonist. These inhibitory effects were blocked by a muscarinic but not by a nicotinic receptor antagonist. In the presence of VIP or IBMX, which raise intracellular cyclic AMP levels and stimulate hormone release, the degree of muscarinic inhibition was increased, but the potency remained unchanged. Similar changes in the secretory rate of prolactin and growth hormone occurred in these and in cell perifusion experiments. These results suggest that the inhibition of hormone secretion from GH3 cells by muscarinic agonists is mediated by a decrease in intracellular cyclic AMP levels.

CALCIUM, CYCLIC AMP AND HORMONE ACTION

It is now well established that the communication of hormonal signals to the interior of the cell is achieved by the generation of second messengers at the cell membrane level. The concept that intracellular regulators or ‘second messengers’ mediate the effects of hormones that themselves do not enter the cell, was proposed originally by Sutherland et al. (1965). Sutherland and his co-workers found that cyclic AMP played a key role in the response of the liver to adrenalin and to glucagon and discovered the enzyme responsible for the formation of cyclic AMP from ATP, adenylate cyclase. Since then a wide variety of hormones have been shown to stimulate adenylate cyclase in cell membranes. The specificity of the response resides in the specificity of the receptor for a particular hormone on the cell surface. The role of calcium as an intracellular regulator has also been known for many years (Rubin, 1982). However, attention has tended to focus on the importance of calcium in excitable cells such as those of nerve and muscle. As the role of cyclic AMP as an intracellular regulator became increasingly apparent, it was also recognized that this nucleotide does not subserve a primary role in the action of a number of hormones. It soon became clear that a distinction between the action of calcium ions in neuro-secretion and excitation/contraction and cyclic AMP in hormone action in non-excitable tissues was invalid. Indeed, in many excitable and in non-excitable systems calcium ions and cyclic AMP can serve as dual interrelated messengers in determining the cellular responsc to a specific stimulus (Rasmussen, 1980). The purpose of this brief review is to outline the importance of cyclic AMP and of calcium as intracellular regulators, to try to describe how they are generated and terminated as signals and to discuss their interrelated roles in cellular regulation.

The regulation of connective tissue metabolism by vasoactive intestinal polypeptide

Immunohistochemical studies have confirmed the innervation of bone with neuropeptidergic neurons containing vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). In this study, we report effects of VIP on connective tissue cell metabolism. VIP stimulated PGE2 production in human articular chondrocytes, human osteoblast-like cells and human synovial cells, however, stromelysin production was unaffected. VIP also stimulated cAMP production in human osteoblast-like cells, but not in human articular chondrocytes or synovial cells. These findings are suggestive of a role of VIP in connective tissue cell metabolism which may contribute to the inflammatory processes of arthritis.