Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic death of bystander cells.

An emerging strategy for cancer gene therapy involves the transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene into tumor cells, rendering them susceptible to the cytotoxic effects of ganciclovir. The observation that HSV-tk-expressing cells can also induce cell death in neighboring cells, which do not express HSV-tk, has been called the bystander effect. Gap junction-mediated transfer of cytotoxic molecules to bystander cells may be an important mechanism of bystander cell death, although others have suggested a role for phagocytosis. In this study, we evaluated the mode of cell death in bystander cells. We detected apoptosis in bystander cells and found that bystander cell death could be inhibited by BCL2 expression. We determined that ganciclovir incubations for 10 h were sufficient to induce cell death in most bystander cells cocultured with HSV-tk-expressing cells. During this period, no phagocytosis was detected, although it was obvious at later stages.

Bcl-2 overexpression abolishes early calcium waving preceding apoptosis in NIH-3T3 murine fibroblasts.

Overexpression of the apoptosis protection gene bcl-2 abolished the earliest response to serum withdrawal in NIH-3T3 murine fibroblasts, i.e., the abrupt cytoplasmic free calcium drop. This phenomenon, also observed in a myeloid cell line, led us to propose this ionic waving as an early apoptotic signal. Its abolition by bcl-2 overexpression suggests that this gene plays a role also on early events "priming" apoptosis.

Apoptosis induction in 32D cells by IL-3 withdrawal is preceded by a drop in the intracellular calcium level.

The 32D murine myeloid cell line is dependent on interleukin-3 (IL-3) for growth in vitro. We show here that IL-3 induces a slow increase in endocellular calcium, which is sizeable two hours after the addition. Withdrawal of the cytokine induces apoptosis, whose classic late events are evident after 16/18 hours, and are preceded by a calcium drop during the first 2/3 hours after IL-3 subtraction. Calcium drop is here proposed as a trigger of the apoptotic process, in agreement with other recently reported findings.

Negative growth control by a novel low M(r) phosphotyrosine protein phosphatase in normal and transformed cells.

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

Overexpression of a synthetic phosphotyrosine protein phosphatase gene inhibits normal and transformed cell growth.

We studied the level of the cytosolic phosphotyrosine protein phosphatase (PTPase) (originally termed low-M(r) acid phosphatase) in normal NIH/3T3 and in v-erbB-transformed fibroblasts. The level of the enzyme, assayed by ELISA, was inversely related to cell proliferation, normally growing cells had less enzyme than their contact-inhibited counterparts and v-erbB transformants had less enzyme than normal NIH/3T3. In order to overexpress the enzyme and study its effects in normal and transformed cells, we transfected a synthetic gene coding for the PTPase in control NIH/3T3 and v-erbB transformants. The overexpressed enzyme was recognized by antibodies raised against the native enzyme and, in cells overexpressing the PTPase, we observed a marked dephosphorylation of tyrosyl residues of cellular proteins. Cell proliferation, in both normal and v-erbB transformants overexpressing the PTPase, was measured. We observed that PTPase overexpression was accompanied by significantly reduced thymidine incorporation in both cell types, either serum-starved or serum-stimulated. The ability of transformed v-erbB cells to grow in soft agar was also markedly decreased by overexpression of the enzyme. Taken together, our results indicate that overexpression of PTPase might interfere with mitogenic signalling pathways in both normal and transformed cells, and propose a role for PTPase in the control of cell proliferation.

Mitogenic signal transduction: a common target for oncogenes that induce resistance to ionizing radiations.

We hypothesized that resistance to ionizing radiations accompanying neoplastic transformation caused by some oncogenes was due to common biochemical pathways affecting the mechanism of mitogenic signal transduction. In order to verify this hypothesis, we studied the formation of mitogenic second messengers in cells transformed by oncogenes that induce radioresistance. We observed an increase of diacylglycerol which activates protein kinase C, an increase of phosphatidylcholine metabolism, with a concomitant decrease of inositol lipid metabolism. Our data show that sensitivity to ionizing radiations was inversely related to the intracellular level of diacylglycerol; study of signalling alterations in spontaneous tumors could provide predictive indications about the responsiveness of neoplasia to radiation therapy.

Heparin inhibits A431 cell growth independently of serum and EGF mitogenic signalling.

In several cell types heparin exerts an antiproliferative action; here we report that heparin inhibited the growth of human epidermoid carcinoma A431 cells. Heparin binding to the cell surface was necessary for growth inhibition; binding was influenced by the molecular weight of heparin. Inhibition of A431 cell proliferation was evident in the presence and in the absence of serum, thus indicating that heparin did not act by binding and 'subtracting' nutrients or other serum factors. In confluent A431 cells, EGF induced DNA synthesis, but heparin did not inhibit this effect; consistently, it did not affect inositol lipid turnover triggered by EGF or bradykinin.

Inhibition of BC3H-1 cell growth by heparin is related to decreased mitogenic signalling.

We examined the effect of heparin and heparin fragments on BC3H-1 muscle cell proliferation. Heparin significantly inhibited BC3H-1 cell growth and this inhibitory effect was related to the ability of heparin to bind to cell surface; low molecular weight heparins were poorly efficient in binding and inhibiting proliferation. Analysis by gel filtration of heparin bound to cell surface showed selective binding of the high molecular weight fraction. Heparin inhibited serum-stimulated incorporation of [3H]thymidine; this effect, however, was only evident when heparin was administered concomitantly with serum. Similarly, heparin inhibited serum-induced inositol lipid turnover only when present with serum. Heparin fragments unable to inhibit cell growth did not affect the metabolism of inositol lipids. Taken together these data suggest that heparin inhibits cell growth by interfering with growth factor-mediated mitogenic signalling.

Signal transduction in EJ-H-ras-transformed cells: de novo synthesis of diacylglycerol and subversion of agonist-stimulated inositol lipid metabolism.

We examined the level of 1,2-diacylglycerol and inositol phosphates in normal and EJ-H-ras-transformed BALB/3T3 fibroblasts by prelabelling the cells with [3H]glycerol, [3H]inositol, [14C]glucose, [14C]arachidonic acid, and [14C]palmitic acid. Steady-state level of inositol phosphates, however, was the same in control and transformed cells. Diacyglycerol labelling by [14C]arachidonic acid was the same in control and transformed cells. Insulin dramatically increased diacylglycerol labeling by [14C]glucose in normal cells, whereas it did not affect ras-transformed fibroblasts. Neurotransmitter-induced inositol lipid turnover was greatly enhanced in ras-transformed cells; conversely, platelet-derived growth factor and thrombin-stimulated normal cells to a greater extent than transformed fibroblasts. Taken together these results suggest that ras transformation may induce multifarious effects on signal transduction: it may cause de novo synthesis of diacylglycerol and subversion of neurotransmitter and growth factor receptor coupling to inositol lipid metabolism.

Oncogenes, protein kinase C, neuronal differentiation and memory.

The present article intends to substantiate the concept that protein kinase C signals are involved in apparently unrelated phenomena at the level of the nervous system, like rapid changes in cell excitability, long-term potentiation, learning, memory, and neuronal differentiation. While the involvement of the protein kinase C signal in memory-associated processes and synaptic plasticity is now supported by a variety of data, increasing evidence is emerging that neuronal differentiation in vitro is dependent on a protein kinase C signal, induced by Nerve Growth Factor, or, alternatively, by the activated oncogenes v-ras or v-src. These differentiative agents, as well as phorbol esters or membrane depolarization, are eventually able to elicit the expression of signal-sensitive genome trans-activators, like the nuclear oncogene c-fos. To propose a unitary model, we have chosen three examples among neuronal functions which imply the activation of protein kinase C: (i) rapid phosphorylation of ion channels by protein kinase C and transient changes in cell excitability; (ii) prolonged activation of the enzyme during long-term potentiation, which is a long-lasting increase in synaptic efficacy and has been related to learning and memory; (iii) the action of some oncogenes (e.g. v-ras and v-src) which act as differentiative agents in vitro through protein kinase C signals. They are an example to illustrate the likely involvement of this enzyme following expression of the cellular proto-oncogenes c-ras and c-src during the physiological steps of differentiation. We propose that the differences in the observed phenomena are due to the different time-windows in which the signal is offered, prolonged, repeated (or self-reinforced) to biological systems differing in potentiality and complexity.

Altered platelet function in cirrhosis of the liver: impairment of inositol lipid and arachidonic acid metabolism in response to agonists.

Hemorrhagic disorders are common in patients with liver cirrhosis and result from several factors including impaired platelet function. We evaluated platelet aggregation and arachidonic acid metabolism in response to standard agonists in platelet-rich plasma from 12 cirrhotic patients with mild impairment of liver function (Child A), 12 patients with severe liver dysfunction (Child B and C) and 12 healthy subjects. Platelet aggregation and thromboxane A2 production were consistently reduced in patients with severe liver impairment. To determine whether the platelet dysfunction is due to an intrinsic platelet defect or a circulating inhibitor, we measured platelet aggregation and thromboxane A2 synthesis on washed platelets in healthy subjects and in Child B and C patients. The aggregating response of washed platelets in response to thrombin, collagen and arachidonic acid was markedly reduced, suggesting an intrinsic platelet defect. The biochemical events underlying platelet aggregation were investigated by prelabeling platelets with [1-14C]arachidonic acid. Thrombin-induced activation of phospholipase C (measured as the release of [1-14C]phosphatidic acid) and phospholipase A2 (measured as the release of [1-14C]arachidonic acid and its metabolites) was greatly impaired in platelets from patients with severe liver impairment. We conclude that in advanced cirrhosis there is a severe reduction in platelet aggregatory response to physiologic agonists due to an intrinsic platelet defect which is related to an impairment of the platelet transmembrane signaling mechanism induced by receptor stimulation.

Morphine withdrawal in vitro: potentiation of agonist-dependent polyphosphoinositide breakdown.

Naloxone (10(-5) -10(-9) M) significantly increased the K+ (30 mM)-induced release of [3H[noradrenaline when it was applied to cortical slices taken from morphine-dependent rats but did not change the release of transmitter when applied to slices prepared from non-dependent animals. Therefore, this preparation was considered suitable to study withdrawal-related events and was used to monitor the agonist-induced changes of phospholipase C activity in the withdrawal state. Noradrenaline (1-100 microM) and carbachol (50-500 microM), when applied to cortical slices preincubated with [3H]inositol or with [32P]orthophosphate, dose dependently increased the formation of labeled inositol phosphates or of phosphatidic acid. This confirmed that noradrenaline and carbachol increase phospholipase C activity. This increase was significantly enhanced by naloxone (10(-6) M) when the slices were taken from dependent animals. The results now reported show for the first time in mammalian tissues that opioid withdrawal is associated with changes of phosphoinositide metabolism.

Modulation of growth of melanoma.

Combined heparin-cortisone treatment induces regression of growth in a variety of murine tumors including melanoma. We injected 92 inbred C 57 b1/6 male mice each with 5 X 10(5) melanoma cells (B16, B16 F1, and B16 A6 lines) with different metastatic potential. Heparin (400 U/ml) and cortisone acetate (250 mg/kg SC injections) were given daily. Control experiments were performed both with the administration of no drugs and with administration of cortisone alone. Plasminogen activator activity, which is notoriously related to tumor growth, was evaluated using fibrin plate technique in 10 fragments taken before and 20 days after the combined heparin-cortisone treatment of B16 F1 and B16 A6 melanomas. The combined heparin-cortisone treatment slowed tumor growth, but no tumour regression was observed. Cutaneous fibrinolytic activity appeared increased in all specimens after the treatment.

GABA-receptor stimulation enhances norepinephrine-induced polyphosphoinositide metabolism in rat hippocampal slices.

The effect of gamma-aminobutyric acid (GABA)receptor stimulation on norepinephrine (NE)-induced metabolism of polyphosphoinositides (PIPs) was studied in rat hippocampal slices. Inositol phosphates (IPs), PIPs, and phosphatidic acid were measured. NE induced formation of IPs and phosphatidic acid in a dose-dependent manner with an EC50 of 4.5 microM. GABA, 3-aminopropanesulphonic acid (3APS) and muscimol did not affect PIPs breakdown, but they strongly increased (greater than 100%) PIPs metabolism induced by 1 microM NE. Their action was antagonized by bicuculline (10 microM). We discuss the implications of these findings in hippocampal neurotransmission.

Point-mutated p21ras couples a muscarinic receptor to calcium channels and polyphosphoinositide hydrolysis.

A high-affinity muscarinic receptor is detectable both in normal 3T3 mouse fibroblasts and in their transformed counterpart obtained by transfection with the oncogene EJ/T24-H-ras. However, only the transformed cell line is responsive to muscarinic agonist carbamylcholine in terms of Ca2+ influx and polyphosphoinositide hydrolysis, whereas the normal cell line is unresponsive. Using a point-mutated p21ras protein and monoclonal antibodies anti-p21ras, we provide evidences that p21ras couples to receptor-operating calcium channels and to polyphosphoinositide hydrolysis a muscarinic receptor which is uncoupled in normal mouse fibroblasts.

Effects of cortisone with and without heparin on angiogenesis induced by prostaglandin E1 and by S180 cells, and on growth of murine transplantable tumours.

Cortisone acetate, locally applied in sustained-release pellets, is effective in inhibiting angiogenesis induced by prostaglandin E1 in the rabbit cornea. The inhibitory effect of cortisone is not increased by addition of heparin. Similar results were obtained with angiogenesis induced by S180 cells. The effects of cortisone with and without heparin were also studied on 5 transplantable murine tumours: 3 variants of B16 melanoma, Lewis lung carcinoma and fibrosarcoma M4. The effect of cortisone in slowing the growth rate of tumours was modestly potentiated by heparin, but no regression of the tumour mass occurred.

Cooperative effect of exogenous heparin-like compounds and secreted glucocorticoid-induced inhibitor on plasminogen activator in 3T3 cell cultures.

Balb/c 3T3 cultures grown in the absence of serum release both plasminogen activator and plasminogen activator inhibitor in the culture medium. Cellular transformation with SV-40 virus increased the level of the activator, whereas dexamethasone increased the level of the inhibitor. Heparin added to the medium potentiated the glucocorticoid-induced inhibitory activity, strongly decreasing or completely abolishing the activity of plasminogen activator. Heparin sulfate showed similar effects to heparin, although at higher concentrations. It is suggested that heparin-like compounds are involved in the regulation of plasminogen activator, acting as inhibitory cofactors.

Glycosaminoglycans and neoplastic transformation.

Two classes of mammalian glycosaminoglycans (GAGs) are distinguished: hyaluronic acid and chondroitin sulphate homopolymers, exhibiting identical repetitive building blocks, versus heparan and dermatan sulphate co-polymers, with differently substituted disaccharidic units. Evidence reported herein suggests that the latter compounds are positive co-factors of matrix assembly promoting the interaction of collagenous and non-collagenous proteins and inhibiting proteolytic activity. Homopolymers, on the contrary, are recognized as negative co-factors impairing the interaction of matrix proteins. The effect of GAGs on matrix assembly influences all the cell periphery because of trans-membrane relationships between submembranous cytoskeletal structures and the outer pericellular matrix. Molecular mechanisms involving GAGs in neoplastic transformation are proposed and discussed.

Co-polymeric glycosaminoglycans in transformed cells. Transformation-dependent changes in the co-polymeric structure of heparan sulphate.

1. Heparan sulphates from normal 3T3 fibroblasts are association-prone as indicated by their affinity for agarose gels substituted with cognate heparan sulphate species. Heparan sulphates from SV40-transformed or polyoma-virus-transformed cells have no affinity for the same gels. 2. Heparan sulphates from the medium, the pericellular and intracellular pools of normal, SV40-transformed and polyoma-transformed 3T3 cells were separated into four subfractions (HS1-HS4) by ion-exchange chromatography. In general, HS1-HS3 were found in cell-derived heparan sulphates, whereas HS3-HS4 were present in the medium. The heparan sulphates from transformed cells were more heterogeneous and of lower charge density than those from the normal counterpart. 3. Degradations via periodate oxidation/alkaline elimination yielded the oligomers glucosamine-(hexuronate-glucosamine)(n)-R with n=1-5 and a large proportion of N-sulphate groups. There was a large contribution of fragments n=4-5 from heparan sulphates of normal cells. These fragments were less common in low-sulphated heparan sulphates of transformed cells. In the case of medium-drived heparan sulphates all species had a low content of fragments n=4-5. 4. The size distribution of (glucuronate-N-acetylglucosamine)(n) regions was assessed after deaminative cleavage. It was broad and ranged from n=1-10 for all heparan sulphate species. In the case of medium-derived heparan sulphates there were distinct differences between normal and transformed cells. In the latter chains the N-acetyl-rich segments were both shorter and longer than in the normal case. The shape of the disaccharide peak was consistent with a lower content of O-sulphate in the heparan sulphates from transformed cells. 5. It was concluded that heparan sulphates from medium or transformed cells exhibit the greatest structural deviation from the normal case. The finding of lower proportions of extended, iduronate/glucuronate-bearing, N-sulphate-rich segments in heparan sulphates of transformed cells was particularly interesting in view of the fact that these elements have been associated with ability to self-interact.