Bhas 42 cell transformation activity of cigarette smoke condensate is modulated by selenium and arsenic.

Cigarette smoking remains a major health risk worldwide. Development of newer tobacco products requires the use of quantitative toxicological assays. Recently, v-Ha-ras transfected BALB/c3T3 (Bhas 42) cell transformation assay was established that simulates the two-stage animal tumorigenesis model and measures tumor initiating and promoting activities of chemicals. The present study was performed to assess the feasibility of using this Bhas 42 cell transformation assay to determine the initiation and promotion activities of cigarette smoke condensate (CSC) and its water soluble fraction. Further, the modulating effects of selenium and arsenic on cigarette smoke-induced cell transformation were investigated. Dimethyl sulfoxide (DMSO) and water extracts of CSC (CSC-D and CSC-W, respectively) were tested at concentrations of 2.5-40 µg mL(-1) in the initiation or promotion assay formats. Initiation protocol of the Bhas 42 assay showed a 3.5-fold increase in transformed foci at 40 µg mL(-1) of CSC-D but not CSC-W. The promotion phase of the assay yielded a robust dose response with CSC-D (2.5-40 µg mL(-1)) and CSC-W (20-40 µg mL(-1)). Preincubation of cells with selenium (100 nM) significantly reduced CSC-induced increase in cell transformation in initiation assay. Co-treatment of cells with a sub-toxic dose of arsenic significantly enhanced cell transformation activity of CSC-D in promotion assay. The results suggest a presence of both water soluble and insoluble tumor promoters in CSC, a role of oxidative stress in CSC-induced cell transformation, and usefulness of Bhas 42 cell transformation assay in comparing tobacco product toxicities and in studying the mechanisms of tobacco carcinogenesis.

Rottlerin: a multifaced regulator of keratinocyte cell cycle.

In this study we showed that Rottlerin (also called Kamala or Mallotoxin), a natural product purified from Mallotus phillippinensis, is a potent suppressor of human keratinocytes (HaCaT cell line) proliferation. Following Rottlerin treatment, Thymidine incorporation into DNA and re-epithelialisation in a scratch wound model was decreased. At the molecular level, Rottlerin hampered the NFkB activation process, causing loss of cyclin D1 and promoting, in a PKCdelta-dependent pathway, ERK activation, which, in turn induced the cell cycle inhibitor p21 Cip1/Kip1. The NFkB-dependent drop in cyclin D1, along with the PKCdelta/ERK-dependent induction of p21 Cip1/Kip1, is responsible for growth arrest. These results open the way to further investigation on the Rottlerin therapeutic potential against keratinocyte hyper-proliferative disorders.

Sodium phenyl butyrate downregulates endothelin-1 expression in cultured human endothelial cells: relevance to sickle-cell disease.

As hydroxyurea (HU), sodium phenyl butyrate (SPB) is known to induce fetal hemoglobin (HbF) expression and thus shows potentials for sickle-cell disease (SCD) treatment. More recently, few studies suggested that endothelial cells (ECs), a major pathophysiological actor of SCD, are also a target of SPB. Here, we show that SPB, as HU, reduces endothelin-1 mRNA expression and peptide release by human ECs in culture. SPB increases VCAM-1 and ICAM-1 mRNAs and soluble ICAM-1 release. Both drugs have a cumulative effect on ICAM-1 expression. We conclude that SPB, as HU, also affects the expression of molecules important to the pathophysiology of SCD, in addition to its effect on HbF. Its potential as an alternative or adjuvant drug in SCD treatment warrants further investigations.

Reduction of invasive potential in K-ras-transformed thyroid cells by restoring of TGF-beta pathway.

Transforming Growth Factor-beta1 (TGF -beta1) is a multifunctional cytokine that regulates a number of cellular processes such as cell growth, differentiation, plasticity, cell motility, adhesiveness, embryogenesis, development and apoptosis through binding to TGF-beta receptors. We have previously demonstrated that K-ras-transformed rat thyroid cells, K10, are resistant to the growth inhibitory action of TGF-beta1, because they show a decreased expression of type II receptor (TbetaRII). Clones obtained transfecting TbetaRII, partially revert their malignant phenotype, showing a reduction in the anchorage-dependent and -independent cell growth and a statistically significant decrease in tumourigenicity with respect to the highly malignant parental cells, both in spontaneous and artificial metastases, when transplanted in athymic nude mice. The purpose of the present work is to elucidate the molecular events involved in the modulation of the tumourigenic potential of K-ras-transformed rat thyroid cells overexpressing TbetaRII. Our data demonstrate that the TbetaRII overexpressed in K-ras-transformed thyroid cell clones is a functional receptor and is essential to restore in these cells behaviour similar to that of control cells. The TbetaRII overexpression is responsible for a strong reduction of adhesive and migratory behaviour of highly malignant K-ras-transformed thyroid cells. These results suggest that the restore of a functional TGF-beta receptor in these cells may be useful for the limitation of tumour spread and dissemination.

[Antimitogenic effect of Pygeum africanum extracts on human prostatic cancer cell lines and explants from benign prostatic hyperplasia]. / Efecto antimitogénico de extractos de Pygeum africanum sobre líneas celulares de cáncer de próstata humana y explantes de hiperplasia benigna de próstata.

OBJECTIVES: To analyze the effect of Pygeum africanum extracts on the in vitro proliferation of human prostate cells. METHODS: Prostate cancer cell lines and benign prostatic hyperplasia derived epithelial cells were cultured and treated with P. africanum extracts. The effect on cell proliferation was monitored by H3-thymidine and bromodeoxyuridine uptake and flow cytometry assays. RESULTS: The incubation with P. africanum extracts, with or without addition of amino acids, significantly and in a dose-dependent manner inhibits the proliferation of prostate cancer derived cells LnCaP, PZ-HPV-7, and CA-HPV-10. In the PZ-HPV-7 cells P. africanum extracts counteract the mitogenic action of EGF and block the transition from G1 to S in the cell cycle. P. africanum extracts also exert a potent antimitogenic action on the epithelial cells derived from benign prostatic hyperplasia explants. CONCLUSION: The ethanolic P. africanum extracts have an antimitogenic effect on prostate cancer cells and benign prostatic hyperplasia epithelial cells. Such effect is associated with the inhibition of the mitogenic action of EGF, and it is accompanied by a decrease of cells entering the S Phase of the cell cycle.

Dermal wound healing properties of redox-active grape seed proanthocyanidins.

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. The wound site is rich in oxidants, such as hydrogen peroxide, mostly contributed by neutrophils and macrophages. We proposed that oxidants in the wound microenvironment support the repair process. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. Previously we have reported that a grape seed proanthycyanidin extract containing 5000 ppm resveratrol (GSPE) potently upregulates oxidant and tumor necrosis factor-alpha inducible VEGF expression in human keratinocytes (Free Radic. Biol. Med. 31:38-42, 2001). Our current objective was to follow up on that finding and test whether GSPE influences dermal wound healing in vivo. First, using a VEGF promoter-driven luciferase reporter construct we observed that the potentiating effect of GSPE on inducible VEGF expression is at the transcriptional level. The reporter assay showed that GSPE alone is able to drive VEGF transcription. Next, two dermal excisional wounds were inflicted on the back of mice and the wounds were left to heal by secondary intention. Topical application of GSPE accelerated wound contraction and closure. GSPE treatment was associated with a more well-defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. GSPE treatment also increased VEGF and tenascin expression in the wound edge tissue. Tissue glutathione oxidation and 4-hydroxynonenal immunostaining results supported that GSPE application enhanced the oxidizing environment at the wound site. Oxidants are known to promote both VEGF as well as tenascin expression. In summary, our current study provides firm evidence to support that topical application of GSPE represents a feasible and productive approach to support dermal wound healing.

Organochlorine pesticides directly regulate gonadotropin-releasing hormone gene expression and biosynthesis in the GT1-7 hypothalamic cell line.

Environmental toxicants profoundly affect growth and developmental processes. In the present study, we hypothesized that hypothalamic gonadotropin-releasing hormone (GnRH) neurons, which regulate the reproductive axis, are targets of environmental endocrine disrupting chemicals. Two organochlorine pesticides (methoxychlor and chlorpyrifos) were tested for their effects on GnRH gene expression and biosynthesis in the immortalized hypothalamic GT1-7 cells, which synthesize and secrete GnRH. GT1-7 cells were treated with methoxychlor or chlorpyrifos for 24 h in dose-response experiments, and GnRH gene expression and peptide levels were quantified. In order to examine whether these pesticides affect GnRH biosynthesis through the estrogen receptor (ER), in other experiments their effects were compared to those of estrogen, or they were co-administered with the ER antagonist, ICI 182,780 (ICI). Both methoxychlor and chlorpyrifos had significant effects on GnRH gene transcription and GnRH mRNA levels. These effects were not consistently blocked by ICI, nor did the effects of these pesticides consistently mimic those of estrogen, suggesting a mechanism independent of the ER. Chlorpyrifos and methoxychlor slightly stimulated peptide levels, and this effect was blocked by ICI, suggesting that the ER may mediate effects of pesticides on GnRH release. These results indicate that chlorpyrifos and methoxychlor alter GnRH biosynthesis in this hypothalamic cell line in vitro, suggesting that they may have endocrine disrupting effects on GnRH neurons in vivo.

The effects of a protease inhibitor fraction from tepary bean (Phaseolus acutifolius) on in vitro cell proliferation and cell adhesion of transformed cells.

Some protease inhibitors (PI), such as the soybean Bowman-Birk protease inhibitor (SBBI), have been described as anticarcinogenic agents. Although PI are ubiquitous compounds in living organisms, the anticarcinogenic potential of PIs other than SBBI remain poorly explored. We evaluated the antiproliferative effect of a protein fraction from tepary bean (Phaseolus acutifolius) seeds with protease inhibitor activity (TPIF), on normal and on malignant cells. TPIF was obtained after precipitation with ammonium sulfate and gel filtration, and its bioactivity was assayed in vitro on HeLa cells, normal 3T3 fibroblasts and 3T3/v-mos transformed fibroblasts. TPIF showed antiproliferative and cytotoxic effects on 3T3/v-mos transformed fibroblasts in a dose-dependent way. On the contrary, TPIF was only cytostatic for normal 3T3 cells at the highest doses assayed, and had no effect on epithelial HeLa cells proliferation. Sublethal TPIF doses also stimulated cell adhesion of poorly adherent 3T3/v-mos cell line.

Potent cytotoxic lignans from Justicia procumbens and their effects on nitric oxide and tumor necrosis factor-alpha production in mouse macrophages.

A new lignan glycoside, 4-O-alpha-L-arabinopyranosyl-(1' "-->2' ')-beta-D-apiofuranosyldiphyllin (2), named procumbenoside A, and 11 known compounds were isolated from the whole plant of Justicia procumbens. The structure of 2 was established by spectral analysis and chemical methods. The known compounds justicidin A (1), diphyllin (3), and tuberculatin (4) showed potent cytotoxic effects against a number of cancer cells in vitro. Compounds 1 and 4 also strongly enhanced tumor-necrosis factor-alpha (TNF-alpha) generation from mouse macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS).

Treatment of spinal muscular atrophy by sodium butyrate.

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, leading to muscular paralysis with muscular atrophy. No effective treatment of this disorder is presently available. Studies of the correlation between disease severity and the amount of survival motor neuron (SMN) protein have shown an inverse relationship. We report that sodium butyrate effectively increases the amount of exon 7-containing SMN protein in SMA lymphoid cell lines by changing the alternative splicing pattern of exon 7 in the SMN2 gene. In vivo, sodium butyrate treatment of SMA-like mice resulted in increased expression of SMN protein in motor neurons of the spinal cord and resulted in significant improvement of SMA clinical symptoms. Oral administration of sodium butyrate to intercrosses of heterozygous pregnant knockout-transgenic SMA-like mice decreased the birth rate of severe types of SMA-like mice, and SMA symptoms were ameliorated for all three types of SMA-like mice. These results suggest that sodium butyrate may be an effective drug for the treatment of human SMA patients.

In vitro anti-HIV activity of sulfated cell-wall polysaccharides from gametic, carposporic and tetrasporic stages of the Mediterranean red alga Asparagopsis armata.

The gametic, carposporic and tetrasporic reproductive stages from the Mediterranean red alga Asparagopsis armata contain peculiar sulfated galactans with galactose:3,6-anhydrogalactose:sulfates molar ratio of 1:0.01:1.23, 1:0.04:0.47 and 1:0.01:1.13, respectively. These water-soluble polysaccharides were studied for their in vitro activity against the human immunodeficiency virus (HIV-1). Gametic and tetrasporic galactans inhibit HIV replication at 10 and 8 micrograms/ml, respectively, as measured by HIV-induced syncitium formation as well as reverse transcriptase activity in cell-free culture supernatant. The carposporic polysaccharide is ineffective, even at 100 micrograms/ml. The maximal antiviral effect involves the presence of the polysaccharides after or during infection but not before infection. This time of action suggests an inhibition of an early step of HIV infection.

ARG tyrosine kinase activity is inhibited by STI571.

The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces hematologic remission in most patients with chronic myeloid leukemia. In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF)beta receptor, and c-KIT, but it does not inhibit SRC family kinases, c-FMS, FLT3, the epidermal growth factor receptor, or multiple other tyrosine kinases. ARG is a widely expressed tyrosine kinase that shares substantial sequence identity with c-ABL in the kinase domain and cooperates with ABL to regulate neurulation in the developing mouse embryo. As described here, ARG has recently been implicated in the pathogenesis of leukemia as a fusion partner of TEL. A TEL/ARG fusion was constructed to determine whether ARG can be inhibited by STI571. When expressed in the factor-dependent murine hematopoietic cell line Ba/F3, the TEL/ARG protein was heavily phosphorylated on tyrosine, increased tyrosine phosphorylation of multiple cellular proteins, and induced factor-independent proliferation. The effects of STI571 on Ba/F3 cells transformed with BCR/ABL, TEL/ABL, TEL/PDGFbetaR, or TEL/ARG were then compared. STI571 inhibited tyrosine phosphorylation and cell growth of Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/PDGFbetaR, and TEL/ARG with an IC(50) of approximately 0.5 microM in each case, but it had no effect on untransformed Ba/F3 cells growing in IL-3 or on Ba/F3 cells transformed by TEL/JAK2. Culture of TEL/ARG-transfected Ba/F3 cells with IL-3 completely prevented STI571-induced apoptosis in these cells, similar to what has been observed with BCR/ABL- or TEL/ABL-transformed cells. These results indicate that ARG is a target of the small molecule, tyrosine kinase inhibitor STI571.

Bemiparin and fluid flow modulate the expression, activity and release of tissue factor pathway inhibitor in human endothelial cells in vitro.

We investigated the localisation, gene expression, and activity of tissue factor pathway inhibitor (TFPI) in endothelial cells (EC) grown in static conditions or under shear stress, in the presence of unfractionated heparin (UFH) and two low-molecular-weight heparins (LMWHs). dalteparin and bemiparin (a second generation of LMWHs). All three preparations induced increased release, cellular redistribution, and enhanced activity of TFPI on the cell surface in static EC. In EC grown under shear stress (0.27, 4.1 and 19 dyne/cm2) and incubated with each heparin for 24 h, the release of TFPI was significantly correlated with the level of flow for bemiparin and dalteparin, but not for UFH. For all three levels of flow tested, bemiparin induced the highest secretion and increase of both cellular TFPI and cell surface activity of the inhibitor. The expression of TFPI mRNA, determined by Northern blotting, was specifically modulated by heparins. All three preparations increased the expression of TFPI by 60 to 120% in EC under minimal flow, but only bemiparin enhanced TFPI mRNA in EC under the arterial flow. Immunogold electron microscopy revealed that EC exhibited strong cellular labelling for TFPI when grown under arterial flow in the presence of bemiparin. We conclude that in EC subjected to shear stress in vitro bemiparin is more efficient than UFH or dalteparin in modulating the expression. release and activity of TFPI. We therefore suggest that bemiparin may be superior over the conventional heparins in maintaining the anticoagulant properties of the endothelium.

Arglabin-DMA, a plant derived sesquiterpene, inhibits farnesyltransferase.

Arglabin [1(R),10(S)-epoxy-5(S),5(S),7(S)-guaia-3(4),11(13)-dien-6, 12-olide], a sesquiterpene gamma-lactone is isolated from Artemisia glabella, a species of wormwood endemic to the Karaganda region of Kazakstan. The compound has been modified to render it water-soluble through addition of a dimethylaminohydrochloride group to the C(13) carbohydride moiety to yield Arglabin-DMA. Arglabin-DMA is a registered antitumor substance in the Republic of Kazakstan. Previously, we have shown that this compound prevents protein farnesylation without altering geranylgeranylation. We now report that Arglabin-DMA inhibits the incorporation of [(3)H]farnesylpyrophosphate into human H-ras protein by FTase with an IC(50) of no greater than 25 microM. Kinetic studies show that the phosphorylated form of this compound competitively inhibits the binding of farnesyl diphosphate to FTase. This mechanism of action is different from other reported peptidomimetic FTIs which lower the affinity of ras protein to FTase. Our in vitro studies confirm that Arglabin-DMA inhibits post-translational modification of ras protein in cells. Arglabin-DMA inhibits anchorage-dependent proliferation of NB cells (IC50=10 microg/ml) and inhibits anchorage-independent growth of NB and KNRK cells with about the same IC(50). Soft-agar colony formation assay of H-ras and K-ras transformed cells show IC(50)s to be 2 and 5 microg/ml, respectively. In primary cultures of human tumor cells, Arglabin-DMA inhibits cell proliferation of a variety of tumor types with IC(90)s in the range of 0.85 to 5.0 microg/ml. Because of these pharmacologic properties, we propose that Arglabin-DMA is suitable for the treatment of ras related malignancies.

Arsenic trioxide induces apoptosis of HPV16 DNA-immortalized human cervical epithelial cells and selectively inhibits viral gene expression.

Arsenic trioxide (As2O3), a major ingredient of arsenic compounds in traditional Chinese medicine, exhibits anti-acute promyelocytic leukemic activity. Considering that over 80% of human malignant tumors derive from epithelial cells, we studied the effect of As2O3 on HPV 16 DNA-immortalized human cervical epithelial cells (HCE16/3 cells) in vitro. As2O3 reduced HCE16/3 cell survival, induced apoptosis at a low concentration and selectively inhibited expression of viral early genes. This effect was evidenced by a reduction of cell viability in the MTT assay, G1 arrest and significant apoptosis upon flow-cytometric analysis, presence of apoptotic bodies, formation of DNA ladders upon gel electrophoresis and inhibition of viral early gene expression by RT-PCR and Western blot. There was a good correlation between cell apoptosis and viral early gene inhibition after As2O3 treatment, suggesting that induction of apoptosis of HCE16/3 cells by As2O3 treatment might be associated with down-regulation of viral oncogene expression. In conclusion, our findings indicate that As2O3 induces apoptosis of HCE16/3 cells, which may provide a new approach for treating HPV-associated tumors.

Fish oil constituent docosahexa-enoic acid selectively inhibits growth of human papillomavirus immortalized keratinocytes.

The omega-3-fatty acids inhibit proliferation of breast cancer cells whereas omega-6-fatty acids stimulate growth. In this study, we examined effects of these fatty acids on human pre-cancerous cells. Cervical keratinocytes, immortalized with the oncogenic human papillomavirus (HPV) type 16, were treated with linoleic acid, an omega-6-fatty acid, and the omega-3-fatty acids, eicosapentaenoic and docosahexaenoic acids. Using both cell counts and bromodeoxyuridine incorporation, docosahexaenoic acid inhibited growth of these cells to a greater extent than eicosapenta-enoic acid. Linoleic acid had no effect. The effect of docosahexaenoic acid was dose dependent and caused growth arrest. Docosahexaenoic acid inhibited growth of HPV16 immortalized foreskin keratinocytes and laryngeal keratinocytes grown from explants of benign tumors caused by papillomavirus, but had no effect on normal foreskin and laryngeal keratinocytes. Docosahexaenoic acid inhibited growth in the presence of estradiol, a growth stimulator for these cells. Indomethacin, a cyclooxygenase inhibitor like docosahexaenoic acid, had only minimal effect on growth. Alpha-tocopherol, a peroxidation inhibitor, abrogated effects of docosahexaenoic acid implying that inhibitory effects were via lipid peroxidation.

Liposome-encapsulated hemoglobin modulates lipopolysaccharide-induced tumor necrosis factor-alpha production in mice.

OBJECTIVE: To investigate the effect of liposome-encapsulated hemoglobin, an experimental blood substitute, on the function of the mononuclear phagocytic system in normovolemic mic, in ex vivo murine splenocytes and in a transformed murine monocytic cell line, RAW 264.7. DESIGN: Prospective, randomized trial. SETTING: Center for Biomolecular Science and Engineering, Naval Research Laboratory, and the Thomas Jefferson University. SUBJECTS: Female Balb/c mice (n = 27). INTERVENTIONS: Mice were injected into the tail vein with liposome-encapsulated hemoglobin or liposome vehicle and were killed at varying time points for blood sampling and splenocyte isolation and culture. MEASUREMENTS AND MAIN RESULTS: Injection of liposome-encapsulated hemoglobin in mice (2.2 of lipid/kg and 0.56 g of hemoglobin/kg, n = 9) did not increase serum tumor necrosis factor (TNF)-alpha concentrations at 2, 8, 15, and 24 hrs after administration. In the ex vivo procedure, lipopolysaccharide (1 microgram/mL)-induced TNF-alpha production by splenocytes from mice injected with liposome-encapsulated hemoglobin was attenuated at 2 and 4 hrs (73%, p = .002 at 2 hrs), compared with TNF-alpha production by splenocytes from sham animals challenged with the same concentration of lipopolysaccharide. In the in vitro procedure, simultaneous exposure of liposome-encapsulated hemoglobin (0.88 to 8.8 mg/mL) and lipopolysaccharide (0.125 to 1 microgram/mL) to the murine-derived, peritoneal monocytic RAW 264.7 cell line showed significantly reduced TNF-alpha peptide, but not messenger RNA, 1 to 4 hrs after exposure as compared with cells challenged with lipopolysaccharide alone. This effect correlated with the rapid phagocytosis (1 hr to 4 hrs) of liposome-encapsulated hemoglobin by RAW 264.7 cells. Phagocytic activity in RAW 264.7 cells exposed to both liposome-encapsulated hemoglobin and lipopolysaccharide showed reduced uptake compared with uptake of liposome-encapsulated hemoglobin. The liposome-induced reduction in TNF-alpha peptide production elicited by lipopolysaccharide was countered by extending the time period to an overnight delay between liposome-encapsulated hemoglobin exposure and lipopolysaccharide challenge. Liposome-encapsulated hemoglobin incubated with lipopolysaccharide in vitro, and subsequently washed to remove free lipopolysaccharide, stimulated TNF-alpha expressed by RAW 264.7 cells. Incubation with liposome-encapsulated hemoglobin alone did not evoke TNF-alpha production in these cells. CONCLUSIONS: These data suggest that liposome-encapsulated hemoglobin modulates the response of the mononuclear phagocyte system to endotoxin, possibly through binding of lipopolysaccharide, presentation to macrophages with subsequent phagocytosis, and modulation of cytokine response by a posttranscriptional mechanism. This effect is attenuated by extending the period between exposure to liposome-encapsulated hemoglobin and endotoxin. The clinical relevance of these findings awaits further investigation.

Intercellular calcium waves in neurons.

Spontaneous intercellular Ca2+ waves were observed in groups of neurons in two different culture preparations: primary mouse cortical neurons and GT1-1 immortalized neurons. Waves of increased intracellular Ca2+ concentration propagated at rates of 100-200 microns/s over as many as 200 cells and were abolished by the removal of extracellular calcium, by nimodipine, by tetrodotoxin, and by the gap junction inhibitor octanol. A sister clone of the GT1 line, GT1-7 neurons, showed no intercellular Ca2+ waves and were found to have a significantly lower level of connexin26 mRNA than the GT1-1 line. Although we cannot definitively rule out a role for synaptic communication, we propose that intercellular Ca2+ waves in cultured neurons are generated by Ca2+ influx caused primarily by the propagation of depolarization via gap junctions. Intercellular Ca2+ signaling via gap junctions may represent an important mechanism for nonsynaptic neuronal signaling.

Uptake, cytotoxicity and metabolism of m-azidopyrimethamine and related lipophilic antifolates in SV-K14 human keratinocytes in vivo.

The growth-inhibitory properties of a series of lipophilic diaminopyrimidine antifolates were evaluated in comparison with methotrexate (MTX) against SV-K14 human keratinocytes in vitro under folate-dependent and folate-independent conditions. Under folate-dependent conditions metoprine (DDMP) proved more cytotoxic than MTX, despite the greater inhibitory activity of the latter compound against mammalian dihydrofolate reductase (DHFR), possibly reflecting differences in cellular accumulation. The significantly lower activity of both compounds under folate-independent conditions indicated DHFR as the primary target. Pyrimethamine (PYM), m-azidopyrimethamine (MZP) and m-aminopyrimethamine (MAP), a metabolite of MZP, were approximately equiactive but less cytotoxic than MTX or DDMP. The unexpected activity of MAP, an inferior DHFR inhibitor, suggests differences in the mechanism of action or cellular transport of the drug, although the reduction of cytotoxicity observed under folate-independent conditions indicate folate metabolism as the cytotoxic locus. In contrast, the cytotoxicity of PYM or MZP was not reduced under folate-independent conditions implying an alternative mechanism of action. The uptake of 2-[14C]pyrimethamine by SV-K14 keratinocytes was rapid with steady-state intracellular concentrations being observed after approximately 100 min, partition of drug into the plasma membrane preceding redistribution and extensive accumulation within the particulate cell components. The previously reported NADPH-dependent metabolism of MZP to MAP by murine liver microsome preparations was not observed with SV-K14 keratinocytes nor with murine skin homogenates in the present study.

The effect of lonidamine (LND) on radiation and thermal responses of human and rodent cell lines.

Rodent and human cells were tested for response to Lonidamine (LND) (1-(2,4 dichlorobenzyl) 1-indazol-3-carboxylic acid) combined with radiation or hyperthermia. Lonidamine exposure before, during, and after irradiation caused varying degrees of inhibition of potentially lethal damage (PLD) repair which was cell line dependent. In human glioma, melanoma, squamous cell carcinoma, and fibroblasts, LND exposure did not inhibit or only partially inhibited repair of potentially lethal damage. LND up to 100 micrograms/ml produced only a low level of toxicity in these cells and only slightly inhibited glucose consumption at the maximum concentration. In human glioma cells, LND treatment alone did not inhibit PLD repair, but when combined with hyperthermia treatment at moderate levels easily achievable in the clinic, there was complete inhibition of potentially lethal damage repair. These data suggest that LND effectiveness is cell type dependent. Combinations of LND, hyperthermia, and radiation may be effective in cancer therapy especially in tumors such as glioma in which repair of potentially lethal damage may be extensive.