Effect of inhibitors of poly(ADP-ribose) polymerase on the induction of GRP78 and subsequent development of resistance to etoposide.

We have recently demonstrated that cell lines deficient in poly(ADP-ribose) synthesis due to deficiency in the enzyme poly(ADP-ribose) polymerase (PADPRP) or depletion of its substrate NAD+ overexpress GRP78. Furthermore, this overexpression of GRP78 is associated with the acquisition of resistance to topoisomerase II-directed drugs such as etoposide (VP-16); (S. Chatterjee et al., Cancer Res., 54: 4405-4411, 1994). Thus, our studies suggest that interference with NAD+-PADPRP metabolism could provide an important approach to (a) define pathways of GRP78 induction, (b) study the effect of GRP78 on other cellular processes, (c) elucidate the mechanism of GRP78-dependent resistance to topoisomerase II targeted drugs, and (d) modulate responses to chemotherapy in normal and tumor tissues. However, in the in vivo situation, it is impractical to interfere with NAD+-PADPRP metabolism by mutational inactivation of PADPRP or by depletion of its substrate NAD+. Therefore, we have examined several inhibitors of NAD+-PADPRP metabolism including 3-aminobenzamide, PD128763, and 6-aminonicotinamide for their ability to reproduce the results obtained with cell lines deficient in NAD+-PADPRP metabolism relative to the induction of GRP78 and subsequent development of resistance to VP-16. Our studies show that 6-aminoicotinamide treatment is highly effective in the induction of GRP78 and subsequent development of resistance to VP-16, whereas treatment with 3-aminobenzamide or PD128763 does not induce GRP78 and thus does not result in VP-16 resistance.

Genetics of susceptibility to 6-aminonicotinamide-induced cleft palate in the mouse: studies in congenic and recombinant inbred strains.

In a search for genetic differences in susceptibility to cleft palate, congenic and recombinant inbred strains of mice were treated with 6-aminonicotinamide or control injections. Of six loci tested, only the chromosome segment marked by N-acetyl transferase was found to affect susceptibility to 6-aminonicotinamide-induced cleft palate. This chromosome segment is known to affect glucocorticoid-induced cleft palate and phenytoin-induced cleft lip with or without cleft palate in these strains of mice.

The effect of 6-aminonicotinamide on energy metabolism in rat embryo neural tube.

6-Aminonicotinamide (6-AN) is a potent metabolic inhibitor which produces central nervous system malformations when administered to pregnant rats. Since the antimetabolite interferes with energy yielding pathways, the present study was undertaken to determine the effect of maternally administered 6-AN on energy metabolism of the neural tube in developing embryos. On day 9 of gestation, pregnant rats were injected with 2 mg/kg 6-AN, and embryos removed 12 or 24 hours later. Glucose, glycogen, lactate, ATP, PCr, and GABA were measured in neural tube sections using fluorometric techniques. Only ATP and PCr showed changes as compared to appropriate controls. These two metabolites were increased 29% and 37%, respectively, by 6-AN administration. The mechanism of these alterations in metabolites is unclear, but may be related to decreased metabolic demand.

Regeneration of retinal pigment epithelium damaged in rat foetuses by 6-aminonicotinamide.

The pathological changes induced by 6-AN (administered i. p. on day 15 of pregnancy) in the pigment epithelium of the retina were studied on days 17, 18, 19 and 20 of pregnancy by light and electronmicroscopy. The marked vacuolar degeneration (enlargement of the perinuclear cisterns) observed until day 18 of pregnancy is followed on days 19 and 20 by a practically total regeneration, by the restitution of the normal microscopic and submicroscopic feature. Within the eye, the regenerative phenomena mentioned are limited to the pigment epithelium (although initially similar pathological changes are observed also in its other components.

Acute nicotinamide deficiency in the pig induced by 6-aminonicotinamide.

Pigs given intraperitoneal injections of 6-aminonicotinamide developed a syndrome similar to a natural disease found in Queensland. Focal lesions, involving mainly destruction of glial cells, developed in the intermediate grey matter of the cervical and lumbar enlargement of the spinal cord. There was widespread vacuolation of spinal cord white matter. The glial cell loss was repaired. We suggest that natural acute nicotinamide deficiency can occur if a threshold dose of an antimetabolite of nicotinamide is absorbed over a short period of time.

Genetics of clefting in the mouse.

Whether the relative sensitivity of A/J to CP induction by many teratogens is due to a common mechanism has not been explored beyond cortisone and 6-AN. These latter were found to be probably independent systems. For 6-NA it was suggested that the strain difference was due to three loci with dominance and epistasis and acting in embryos, so that relative liability like that shown by A/J would be derived from simultaneous homozygosity for recessive genes at three loci. The strain difference in CP response to cortisone was explained by two loci with dominance, but with independent effects (no epistasis), and a maternal effect. At least part of the maternal effect and one of the embryonic loci were associated with the H-2 complex.

Topical 6-aminonicotinamide plus oral niacinamide therapy for psoriasis.

Ninety-nine patients with psoriasis were treated topically with 6-aminonicotinamide (6-AN) in four years. In a double-blind study, 1% 6-AN gel was superior to 0.1% triamcinolone acetonide in 29 comparisons, equal in three, and inferior in one when applied without occlusion for four weeks. In an open study, 1% 6-AN was better than four "potent" steroid creams in 34 comparisons, equal in four, and inferior in one. Substantial improvement or complete clearing of plaques occurred in 85 of 99 patients. Tachyphylaxis occurred in ten, but was not permanent. Mucocutaneous toxicity appeared in 25% but was usually easily controlled. Tinnitus occurred in four, but none had deterioration of audiogram readings. The combination of topical 6-AN and oral niacinamide therapy gives promise of an effective and safe treatment for psoriasis. One of three patients with pityriasis rubra pilaris improved considerably with 6-AN.

6-Aminonicotinamide-induced eye defects in rats.

The pathological changes and structural anomalies induced by 6-aminonicotinamide (6-AN) in the developing eye were studied in rats (Wistar and hooded randombred strain). The substance was administered in aqueous solution intraperitoneally (4 mg/kg on day 9--10 of pregnancy: 5 mg/kg on day 11 of pregnancy; 8 mg/kg on day 13--17 of pregnancy) and in physiological saline intraamniotically (0.01 ml of a 1% solution in physiological saline on day 15 of pregnancy). Embryos and foetuses from experimental series and from untreated control series were macro- and microscopically examined on day 10--20 of pregnancy. Control foetuses from mothers injected with distilled water on day 9--17 of pregnancy were examined on day 20 of pregnancy. The pathological changes and structural anomalies detected at successive developmental stages are presented. They reveal an obvious phase specificity and attest that the same substance may act through both of the main teratogenic pathways hypothetically put forward by Menkes et al. (1970). Based upon the present findings (and some previous results obtained in experiments with bisazo dyes) a working hypothesis is tentatively presented, as to the possible determination of the uni- or/and bilateral distribution of chemically induced developmental defects. In connection with some reversible or transitory pathological changes the role of recovery in teratogenesis is pointed out.

Repression of glial RNA transcription during the development of 6-aminonic-otinamide (6-AN)-induced acute gliopathy.

Administration of 6-aminonicotinamide (6-AN) to rats leads to an opposite effect on the rate at which [3H]UMP is incorporated in vitro into nuclear cerebral neuronal and glial RNA. The inhibition of glial RNA synthesis is temporarily accompanied by both a reduction of the number of RNA initiation sites on glial chromatin and a reduction of [3H]acetate uptake into glial chromatin-bound histones mainly as regards the fraction H2B, H3 and H4. The slight stimulation of neuronal RNA synthesis 6 hours after 6-AN seemed to be caused exclusively by a less steric restriction of neuronal chromatin, whereas the more pronounced stimulation at 24 hours may be related to both a higher activity and/or amount of endogenous neuronal RNA polymerases and an increase in the total number of RNA initiation sites present on the neuronal chromatin. The increase in the total number of neuronal RNA initiation sites is closely parallel with a higher degree of acetylation of neuronal chromatin-bound histone fractions. The 6-AN-induced variations of glial and neuronal in vitro RNA synthesis are discussed in correlation with the neurotoxic action of 6-AN in vivo.