Light- and electron microscopy studies on the cerebellum in 12-day-old rats after treatment with 6-aminonicotinamide (6-AN)].

Light- and electron-microscopie studies were performed in glia cells and neurons of the cerebellum of 12-day-old rats until 48 following intraperitoneal application of 8 mg/kg 6-aminonicotinamide (6-AN), an antimetabolite of nicotinamide that inhibits the oxidative pentose-phosphate pathway. Particularly sensitive were the neuroblasts of the external granular layer. The migration of the neuroblasts was clearly reduced. Parts of the internal granular layer were damaged, while the Purkinje cells and astrocytes remained unchanged. Oligodendrocytes and astrocytes showed vacuolar degeneration. Swelling of the myelin sheath occurred in the form of a status spongiosus. 48 h after 6-AN injection some mitoses and phagocytic cells were found in the internal granular layer.     

Thiol oxidation inhibits nitric oxide-mediated pulmonary artery relaxation and guanylate cyclase stimulation

The mechanisms through which thiol oxidation and cellular redox influence the regulation of soluble guanylate cyclase (sGC) are poorly understood. This study investigated whether promoting thiol oxidation via inhibition of NADPH generation by the pentose phosphate pathway (PPP) with 1 mM 6-aminonicotinamide (6-AN) or the thiol oxidant diamide (1 mM) alters sGC activity and cGMP-associated relaxation to nitric oxide (NO) donors [S-nitroso-N-acetylpenicillamine (SNAP) and spermine-NONOate]. Diamide and 6-AN inhibited NO-elicited relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and stimulation of sGC activity in BPA homogenates. Treatment of BPA with the thiol reductant DTT (1 mM) reversed inhibition of NO-mediated relaxation and sGC stimulation by 6-AN. The increase in cGMP protein kinase-associated phosphorylation of vasodilator-stimulated phosphoprotein on Ser239 elicited by 10 microM SNAP was also inhibited by diamide. Activation of sGC by SNAP was attenuated by low micromolar concentrations of GSSG in concentrated, but not dilute, homogenates of BPA, suggesting that an enzymatic process contributes to the actions of GSSG. Relaxation to agents that function through cAMP (forskolin and isoproterenol) was not altered by inhibition of the pentose phosphate pathway or diamide. Thus a thiol oxidation mechanism controlled by the regulation of thiol redox by NADPH generated via the pentose phosphate pathway appears to inhibit sGC activation and cGMP-mediated relaxation by NO in a manner consistent with its function as an important physiological redox-mediated regulator of vascular function.     

Glucose 6-phosphate dehydrogenase inhibition sensitizes melanoma cells to metformin treatment

Most cancer cells exacerbate the pentose phosphate pathway (PPP) to enhance biosynthetic precursors and antioxidant defenses. Metformin, which is used as a first-line oral drug for the treatment of type 2 diabetes, has been proposed to inhibit the malignant progression of different types of cancers. However, metformin has shown poor efficacy as single agent in several clinical trials. Thus, the aim of the present work was to investigate whether the pharmacological inhibition of G6PDH, the first and rate-limiting enzyme of the PPP, by 6-amino nicotinamide (6-AN) potentiates the antitumoral activity of metformin on different human melanoma cell lines. Our results showed that 6-AN has sensitizing properties to metformin cytotoxicity. The combination of metformin and 6-AN decreased glucose consumption and lactate production, altered the mitochondrial potential and redox balance, and thereby blocked melanoma cell progression, directing cells to apoptosis and necrosis. To our knowledge, this is the first study describing the effect of this combination. Future preclinical studies should be performed to reveal the biological relevance of this finding.     

Lineage of radial glia in the chicken optic tectum.

In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following. (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor. (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (approximately E5) and after radial migration of immature neurons has begun (E6-7). Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce. (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives. (d) The population of labelled radial glia declines between E15 and E19-20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.     

METABOLITES AND ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN ISOLATED NERVE ENDINGS1

Abstract— The activities of each enzyme associated with the pentose phosphate pathway as well as the non-enzymatic intermediates in this pathway were measured in synaptosomes isolated from rat cerebral cortex. The specific activities of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) were significantly lower in synaptosomes than cerebral cortex; however, the specific activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribosephosphate isomerase (EC 5.3.1.6) and ribulosephosphate epimerase (EC 5.1.3.1.) were comparable in homogenates of synaptosomal fractions and cerebral cortex. Concentrations of most intermediates of the pentose pathway were also similar in extracts of synaptosomes and brain homogenates. Six hours after treatment of rats with the nicotinamide analog, 6-aminonicotinamide (6-AN), 6-phosphogluconate levels in synaptosomes were increased 5-fold; however, glucose-6-phosphate levels remained unchanged. During a 30 min in uitro incubation 6-phosphogluconate levels increased approx 2-fold in synaptosomes obtained from 6-AN treated rats but did not change in synaptosomes from untreated rats. During the same period glucose-6-phosphate levels decreased in synaptosomes from both control and 6-AN treated rats. The conversion of both [1-¹⁴C]glucose and [6-¹⁴C]glucose to ¹⁴CO₂ was depressed in synaptosomes from 6-AN treated rats; however, the ratio of the two isotopes converted to ¹⁴CO₂ was essentially the same. It is concluded that the pentose phosphate pathway is active in nerve endings both in vivo and in vitro.     

Pentose phosphate pathway coordinates multiple redox-controlled relaxing mechanisms in bovine coronary arteries

Pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN) and epiandrosterone (Epi), were employed to examine whether changes in NADP(H) redox regulates contractile force in endothelium-removed bovine coronary arteries (BCAs). 6-AN (0.01-5 mM) or Epi (1-500 microM) elicited dose-dependent relaxation in BCAs contracted with 30 mM KCl, 0.1 microM U-44619, and endothelin-1 but not with phorbol 12,13-dibutyrate, a protein kinase C activator that causes Ca2+-independent contraction. Relaxation to PPP inhibition was associated with oxidation of NADPH and glutathione (GSH). Relaxation to 6-AN was not mediated by H2O2, because it was not altered by hypoxia or the peroxide scavenger ebselen (100 microM). The thiol reductant DTT (3 mM) attenuated the relaxation to 6-AN and Epi by 30-40%. Inhibition of glycolysis or mitochondrial electron transport did not elicit relaxation in BCAs contracted with 30 mM KCl, suggesting these pathways may not be involved in relaxation elicited by PPP inhibition. High doses of K+ channel blockers [e.g., TEA (10 mM) and 4-aminopyridine (10 mM)] only partially inhibited the relaxation to 6-AN. On the basis of changes in the fura-2 fluorescence ratio, 6-AN and Epi appeared to markedly reduce intracellular Ca2+. Thus PPP inhibition oxidizes NADPH and GSH and appears to activate a novel coordination of redox-controlled relaxing mechanisms in BCAs mediated primarily through decreasing intracellular Ca2+.     

Radiosensitization by 2-deoxy-D-glucose and 6-aminonicotinamide involves activation of redox sensitive ASK1-JNK/p38MAPK signaling in head and neck cancer cells

Our previous studies on simultaneous inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) and pentose phosphate activity by 6-aminonicotinamide (6-AN) have been shown to induce oxidative stress mediated selective radiosensitization in wide range of human malignant cells. However, the mechanism of radiosensitization induced by this combination (2-DG+6-AN) is not completely understood. Since activation of apoptotic signal regulating kinase (ASK1) and subsequent apoptosis are implicated in oxidative stress response, the role of ASK1 activation in radiosensitization by this combination was investigated in the present study. Our results demonstrated that redox alterations induced by this combination activated ASK1 and subsequent apoptosis during radiosensitization of head and neck carcinoma cells (KB). In addition, mRNA and protein expression of thioredoxin and thioredoxin reductase decreased significantly under similar treatment conditions. Further, the downstream targets such as JNK and p38MAPK were also activated by this combination, and their pharmacological inhibition by SP600125 and SB201291 respectively resulted in suppression of 2-DG+6-AN mediated apoptosis in irradiated KB cells. Interestingly, the activation of ASK1 was mediated by hydrogen peroxide rather than superoxide anions as PEG-catalase but not PEG-SOD suppressed its activation. Our observations clearly suggest that redox alterations by inhibition of glucose metabolism serves as a molecular switch that activate ASK1-JNK/p38MAPK signaling in malignant cells during radiosensitization by 2-DG+6-AN. The present study emphasizes the importance of redox alterations in determining radiosensitivity of tumor cells that may greatly influence the outcome of radiation therapy.     

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.     

Glucose is necessary for stabilization of hypoxia-inducible factor-1α under hypoxia: Contribution of the pentose phosphate pathway to this stabilization

In this study, we observed that low glucose or fructose reduces the increase in hypoxia-inducible factor-1α (HIF-1α) protein under hypoxic conditions. 6-Aminonicotinamide (6-AN), an inhibitor of the pentose phosphate pathway (PPP), also inhibited the increase of HIF-1α protein under hypoxic conditions, while the reduced protein levels of HIF-1α by low glucose were apparently recovered by the addition of MG-132 or NADPH. Moreover, siRNA for glucose-6-phosphate dehydrogenase, which produces NADPH, reduced the increase in HIF-1α protein. On the other hand, cobalt-induced expression of HIF-1α protein was not affected by low glucose or 6-AN under normoxic conditions. In conclusion, glucose metabolism through the PPP, but not in glycolysis, plays an important role in the stabilization of HIF-1α protein under hypoxic conditions.     

Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium

The objective was to examine potential roles of glucose and pyruvate in nuclear and cytoplasmic maturation of porcine oocytes. Oocyte-cumulus complexes (OCC), derived from 3 to 6mm follicles, were cultured in a chemically defined medium (pyruvate-free mNCSU37-PVA), with or without 5.55mM glucose, during in vitro maturation (IVM); in the absence of glucose, germinal vesicle breakdown (GVBD) and nuclear maturation were prevented (P<0.05). Subsequently, OCC were cultured for IVM in glucose-containing mNCSU37-PVA supplemented with 6-amononicotinamide (6-AN) and diphenyleneiodonium (DPI), inhibitors of the pentose phosphate pathway (PPP); both compounds (>/=10muM 6-AN and >/=10nM DPI) inhibited resumption of meiosis (P<0.05). Supplementation of glucose-free maturation medium with increasing concentrations of pyruvate induced resumption of meiosis and increased the incidence of oocytes reaching metaphase-II in a concentration-dependent manner (P<0.05). More mature oocytes were obtained in the presence of pyruvate+glucose (P<0.05). After culture to allow maturation, glutathione content was higher in oocytes cultured in the presence of pyruvate alone than in those cultured in glucose alone; inclusion of 6-AN abolished responses to pyruvate (P<0.05). In conclusion, both glucose and pyruvate played a critical role in the release of porcine oocytes from arrest at the GV-I stage, probably through the PPP, whereas supplementation with pyruvate improved cytoplasmic maturation, as determined by oocyte glutathione content.     

Affinity of intramitochondrial granules for ruthenium red accompanying induced cell death in chick embryos.

To test the hypothesis that ruthenium red binding of intramitochondrial granules might reflect an altered or pathological state of membranes associated with degeneration, embryos were treated with 6-AN to induce cell death in cartilaginous skeletons of chick embryos. Cervical cartilage from normal, 6-AN-treated and nicotinamide-alleviated 6-AN embryos was examined ultrastructurally for presence of IM RR-positive granules. Mitochondria of normal cervical chondroblasts which undergo normal phenotypic expression acquire RR-positive granules, although few mature cells are observed in young embryos. Necrotic chondroblasts, chondroblasts in various stages of degeneration, and proliferating chondrogenic cells of 6-AN-treated embryos all demonstrated induced RR-positive IM granules. Foci of degenerating chondroblasts, with mitochondria demonstrating RR granules, were observed infrequently in teratogen-alleviated tissue. The cytological features induced by 6-AN confirm its lethal effect and the degenerative effect on membranes presumably "unmasks" mitochondrial Ca-affinity sites which then become RR-positive. Cytochemical observations correspond with the biochemical and structural changes induced by 6-AN and confirm the hypothesis that RR-positive sites are the result of pathological changes.     

Contribution of oxidative stress to radiosensitization by a combination of 2-DG and 6-AN in human cancer cell line

In the present studies, the role of oxidative stress in radiosensitization by a combination of 2-DG and 6-aminonicotinamide (6-AN) was examined in a human glioma cell line (BMG-1: wild type p53). Presence of 2-DG or 6-AN for 4 hr after irradiation (gamma ray 2.5 Gy) significantly enhanced the radiation-induced cell death by 18% and the combination (2-DG + 6-AN) enhanced the cell death by 35%. Neither 2-DG nor 6-AN had any further significant effect on the glutathione levels in irradiated cells. However, the combination (2-DG + 6-AN) caused a significant decrease in GSH content, increase in GSSG levels, and enhanced the superoxide radical generation under these conditions. The enhanced cell death caused by the combination (2-DG + 6-AN) mainly resulted by the process of apoptosis as revealed by annexin V binding and was associated with elevated levels of Cyclin B1. However, no significant change was observed in the levels of Bcl-2. Thus, for the first time, our results have demonstrated that the radiosensitizing effects of these modifiers could also be mediated through alterations in the oxidative stress besides energy limited inhibition of repair and recovery processes.     

Effects of oxidative stress and inhibitors of the pentose phosphate pathway on sexually dimorphic production of IFN-tau by bovine blastocysts

Bovine interferon-tau (IFN-tau), the anti-luteolytic factor secreted by conceptuses of pecoran ruminants, is a product of autosomal genes, yet in vitro produced (IVP) female expanded blastocysts (EB) secrete about twice as much IFN-tau as males. Two possible explanations have been tested here. One is that embryos of one sex are differentially susceptible to oxidative stress. The second is that female EB produce more IFN-tau because pentose-phosphate pathway (PPP) activity is elevated as a result of delayed X-chromosome inactivation. IVP bovine zygotes were cultured to the 8-cell stage and placed under conditions designed either to promote oxidative stress (+/-H2O2; 20 vs. 5% O2), or to inhibit glucose 6-phosphate dehydrogenase (G6PDH) activity (addition of dehydroepiandrosterone, DHEA or 6-aminonicotinamide, 6-AN to the medium). At day 8, blastocysts were cultured individually for a further 48 hr to assess IFN-tau production, and embryo sex determined retrospectively. Blastocyst numbers were reduced (P < 0.05) and their continued development impaired (P < 0.05) in presence of H2O2 (200 microM) and 20% O2, but neither IFN-tau production nor sexually dimorphic expression of IFN-tau were affected. IFN-tau production was reduced, particularly in females (P < 0.05), and sexual dimorphic differences in production were lost in the presence of both DHEA (100 microM) and 6-AN (1 microM). In the case of 6-AN, these effects were achieved without a significant decline in blastocyst developmental progression, quality, or cell number. The data suggest that the higher production of IFN-tau by female EB is an indirect outcome of the increased activity of the oxidative arm of the PPP pathway.     

2-Deoxy-D-glucose and 6-aminonicotinamide-mediated Nrf2 down regulation leads to radiosensitization of malignant cells via abrogation of GSH-mediated defense

Enhanced level of nuclear erythroid-related factor-2 (Nrf2) has been associated with cancer chemo/radioresistance. Therefore, the role of Nrf2 in radiosensitization of malignant cells induced by a combination of 2-deoxy-D-Glucose (2-DG) and 6-aminonicotinamide (6-AN) was investigated. Two established human malignant cells lines namely KB (head and neck squamous carcinoma) and BMG-1 (cerebral glioma) were used. Following treatment with a combination of 2-DG (5 mM) and 6-AN (5 μM), irradiated (2Gy) KB and BMG-1 cells were assessed for protein level of Nrf2, Keap1 and γ-glutamylcysteine synthetase (γ-GCS) by western blotting and mRNA expression of γ-GCS, glutathione reductase (GR) and glutathione peroxidase (GPx1) by RT-PCR at 24 hours post treatment. A significant decrease in the level of Nrf2 with a concomitant increase in Keap1 was observed in both the irradiated malignant cells at 24 hours following treatment with combination (2-DG + 6-AN). Down regulation of γ-GCS, GR and GPx1 at 24 hours following treatment with combination (2-DG + 6-AN) resulted in abrogation of glutathione (GSH)-mediated defense in both the irradiated malignant cells. Eventual accumulation of ROS led to radiosensitization of both the malignant cells. These results indicate that deregulated Nrf2-Keap1 signalling leads to the radiosensitization of malignant cells due to abrogated glutathione defense. Metabolic modification-mediated down regulation of Nfr2 and its downstream signalling may have a potential of improving tumour radiotherapy.     

Correlation between mandibular retrognathia and induction of cleft palate with 6-aminonicotinamide in the rat.

A single injection of the niacin antimetabolite 6-aminonicotinamide (6-AN) late in gestation produces cleft palate in the rat. In order to achieve an understanding of the mechanism of induction of cleft palate, craniofacial growth and palate development were studied in Sprague-Dawley rats after treatment with 6-AN on day 15 of gestation. The rats were maintained on a high niacin diet (95 ppm) and subjected to three different teratogenic levels of 6-AN. The first group was injected with 8 mg/kg, the second was fasted and injected with 8 mg/kg and the third was treated with 16 mg/kg. The lowest teratogenic dose, 8 mg/kg, produced mild mandibular retrognathia on day 16, delayed shelf elevation a few hours and resulted in small rostral and small caudal clefts of the secondary palate. The moderate dose, 8 mg/kg with fasting, produced more severe mandibular retrognathia, delayed shelf elevation about 24 hours and resulted in 37% full clefts and 63% partial clefts of the palate. The highest teratogenic dose, 16 mg/kg, produced severe mandibular retrognathia, delayed shelf elevation by more than 24 hours and resulted in 100% full clefts of the palate. In each 6-AN group, the most severe mandibular retrognathia was present between days 16 and 17, the critical time for palate closure in the rat. Treatment with 6-AN also produced abnormality of the epithelial cells of the palate, the toothbuds and the nasal septum. Molar and incisor toothbuds were small and malformed, and the epithelial surfaces of the palate and the soft tissue nasal septum did not fuse.     

Early changes in parathyroid hormone response and proteoglycan synthesis of chick embryonic femur produced by exposure to 6-aminonicotinamide in ovo

The mechanism of induction of micromelia in 6-day-old chick embryo by 6-aminonicotinamide (6-AN) was investigated. Six-day-old chick embryo exposed to 6-AN did not show micromelia when tenfold excess of nicotinamide over 6-AN was co-administered. The ability of nicotinamide to prevent the induction of micromelia was partially offset after 4 hr of exposure to 6-AN and completely disappeared after 6 hr. The length of time necessary for the induction of micromelia was not affected by the concentration of 6-AN. These results indicate that exposure to 6-AN for only a short period of 6 hr is sufficient to commit the limb to micromelia and that cellular components involved in the induction of micromelia alter during this period. During this period, newly synthesized proteoglycan monomers typical of cartilage decreased in average molecular size, and isolated femora did not respond to parathyroid hormone (PTH) but to dibutyryl cyclic AMP to stimulate growth of cartilage in organ culture.     

ATR signaling mediates an S-phase checkpoint after inhibition of poly(ADP-ribose) polymerase activity

Human fibroblasts, capable of expressing a kinase-dead form of ATR (ATRkd), can be sensitized to the cytotoxic effects of methyl methanesulfonate (MMS) by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN). The combination of MMS+4-AN results in accumulation of cells in S-phase of the cell cycle and activation of Chk1. Inhibition of ATR activity by expression of ATRkd suppresses the S-phase accumulation and partially reverses the Chk1 phosphorylation. The results confirm involvement of an ATR-mediated damage response pathway in the MMS+4-AN-induced S-phase cell cycle checkpoint in human fibroblasts. Consistent with this hypothesis, the inhibitors caffeine and UCN-01 also abrogate the ATR- and Chk1-mediated delay in progression through S-phase. In the absence of ATR-mediated signaling, MMS+4-AN exposure results in a G(2)/M arrest, rather than an S-phase checkpoint. Thus, whereas ATR mediates the S-phase response, it is not critical for arrest of cells in G(2)/M.     

Time of onset and selective response of chondrogenic core of 5-day chick limb after treatment with 6-aminonicotinamide.

Exposure of the chick embryo to the nicotinamide analog, 6-aminonicotinamide (6-AN), causes specific changes in chondrogenic cells that result in limb deformity. Autoradiography has further delineated these changes and relates them to altered utilization of molecular precursors of cartilage matrix and DNA. With ³⁵SO₄ to monitor synthesis of glycosaminoglycan, it was shown that, at 6 hr and persisting until 24 hr after treatment, 6-AN inhibited utilization of sulfate by cells in the chondrogenic core while having no detectable effect on cells in the chondrogenic periphery. Similarly, 6-AN suppressed incorporation of [³H]thymidine into core cells while having no effect to a slight enhancement effect on chondrogenic and nonchondrogenic cells surrounding the core. These observations support the view that, in response to 6-AN-inhibited NAD(P)-dependent reactions, limb chondrogenic cells (CORE) cease to produce matrix glycosaminoglycan, cease to synthesize DNA, and ultimately succumb. Conversely, presumably as a result of more efficient energy production because they lie closer to a vascular supply of oxygen, cells in the chondrogenic periphery withstand the teratogenic insult and continue proliferating to become the source where subsequent partial repair of the limb occurs.     

Migratory paths and phenotypic choices of clonally related cells in the avian optic tectum.

We used retrovirus-mediated gene transfer to study the migration of clonally related cells in the developing chicken optic tectum. Clonal cohorts initially form radial arrays in the ventricular zone (approximately E5), but eventually divide into three separate migratory streams. In the first migration, a minor population of cells migrates tangentially along axon fascicles in medio-laterally directed files (approximately E6-E7); these eventually differentiate into multipolar efferent cells. After E7, the majority of cells in each clone migrate radially along fascicles of radial glia to form the tectal plate, wherein they differentiate into neurons and astrocytes. Around E9, a set of small cells leaves the radial arrays in superficial layers to form a second tangential migration; at least some of these differentiate into astrocytes. Thus, as the tectum develops, cells derived from a single multipotential precursor migrate along three separate pathways, follow separate guidance cues, and adopt distinct phenotypes.     

Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr 78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phenomena also occur in V79-derived cell lines that are deficient in poly(ADP-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitivity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have also previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearing L1210 tumors. These observations prompted us to examine whether 6-AN pre-treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitization to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6-AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immunoblotting. To determine sensitivity after GRP78 up-regulation, the cells were washed and grown for 412 h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412 h time period allowed p(ADPR) metabolism to return to normal while GRP78 levels remained elevated, thus allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, cell sensitivity was determined by clonogenic survival assay or a fluorescence-based cytotoxicity assay. Based on changes in IC50 values, 6-AN caused an increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-expression of GRP78 in human tumor cell lines is associated with increased sensitivity to clinically useful chemotherapy agents. This sensitization occurred in three different tumor cell lines, each bearing a separate genetic defect associated with altered sensitivity.