Wayne S. Stanley

Lignoceroyl-CoASH ligase: enzyme defect in fatty acid β-oxidation system in X-linked childhood adrenoleukodystrophy

We have previously reported that the peroxisomal β‐oxidation system for very long chain fatty acids is defective in X‐linked childhood adrenoleukodystrophy [(1984) Proc. Natl. Acad. Sci. USA 81, 4203‐4207]. In order to elucidate the specific enzyme defect, we examined the oxidation of [1‐14C]lignoceric acid, [1‐14C]lignoceroyl‐CoA and (1‐14C)‐labelled α,β‐unsaturated lignoceroyl‐CoA (substrates for the 1st, 2nd, and 3rd steps of the β‐oxidation cycle, respectively). These studies suggest that the pathognomonic accumulation of very long chain fatty acids in X‐linked childhood ALD may be due to the defective activity of peroxisomal very long chain (lignoceroyl‐CoA) acyl‐CoA ligase.

Adrenoleukodystrophy: impaired oxidation of fatty acids due to peroxisomal lignoceroyl-CoA ligase deficiency.

Very long chain fatty acids (lignoceric acid) are oxidized in peroxisomes and pathognomonic amounts of these fatty acids accumulate in X-adrenoleukodystrophy (X-ALD) due to a defect in their oxidation. However, in cellular homogenates from X-ALD cells, lignoceric acid is oxidized at a rate of 38% of control cells. Therefore, to identify the source of this residual activity we raised antibody to palmitoyl-CoA ligase and examined its effect on the activation and oxidation of palmitic and lignoceric acids in isolated peroxisomes from control and X-ALD fibroblasts. The normalization of peroxisomal lignoceric acid oxidation in the presence of exogenously added acyl-CoA ligases and along with the complete inhibition of activation and oxidation of palmitic and lignoceric acids in peroxisomes from X-ALD by antibody to palmitoyl-CoA ligase provides direct evidence that lignoceroyl-CoA ligase is deficient in X-ALD and demonstrates that the residual activity for the oxidation of lignoceric acid was derived from the activation of lignoceric acid by peroxisomal palmitoyl-CoA ligase. This antibody inhibited the activation and oxidation of palmitic acid but had little effect on these activities for lignoceric acid in peroxisomes from control cells. Furthermore, these data provide evidence that peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases are two different enzymes.

Infantile Fibrosarcoma—a Misnomer?

Two cases of congenital or infantile fibrosarcoma are described that were incompletely excised at the time of primary excision and have not recurred or metastasized after 3 years. The tumors were composed of densely cellular spindle cells with a high mitotic index. Immunohistochemical stains were positive for vimentin but negative for desmin and S-100. The tumor cells were grown in vitro, and a karyotype was obtained. Both tumors had normal diploid modal karyotypes. In addition, fragments of the primary tumor from both cases were injected subcutaneously into nude mice; neither tumor could be heterotransplanted. The clinical course and biologic features of these two tumors suggest that congenital or infantile sarcoma does not have the properties of a malignant neoplasm, and thus the designation of these tumors as a sarcoma may be a misnomer.

Effect of differentiating agents on nucleolar organizer region activity in human melanoma cells

A human cell line established from a metastatic melanoma had both multiple numerical and structural chromosome aberrations including one to two copies of a submetacentric marker chromosome with an insertion of an active nucleolar organizer region (NOR). Treatment of this cell line with retinoic acid (RA) induced morphologic differentiation and reduced the cellular saturation density concomitant with a significant decrease in Ag-NOR activity. RA-treated cells grown in the absence of this differentiating agent, however, displayed a return to normal Ag-NOR activity, indicating the effect of this chemical on ribosomal genes is reversible.

Epidermal growth factor receptor expression in a retinoic acid-treated human melanoma cell line

Treatment of a human cell line (HXG-2), established from a metastatic melanoma, with retinoic acid (RA) induced morphologic differentiation and eliminated its cloning capacity in soft agar. With the v-erb B oncogene as a probe, slot blot hybridization of genomic DNA from parental HXG-2 cells did not show epidermal growth factor (EGF) receptor gene amplification as compared with normal diploid fibroblasts. Analysis of RNA as well as EGF receptor determinations from HXG-2 and RA-treated HXG-2 cells showed essentially no differences, indicating that RA treatment does not modulate EGF receptor gene expression. Although enhanced EGF receptor expression is found in some advanced-stage melanomas, RA-induced changes in the transformation phenotype of cell line HXG-2 probably do not result from modulation of the EGF-mediated pathway.

Isochromosome 11q in a case of Ewing's sarcoma.

A low passage Ewings sarcoma cell line established from a metastatic lesion was cytogenetically analyzed. The modal karyotype was 44,X, -8,i(11q), -15, +12. Other cells had random chromosome aneuploidy superimposed on this karyotype. No cell had a structural rearrangement involving 11q24 or 22q12 as described in other patients with Ewings sarcoma, but all cells had an isochromosome 11q.

In situ genetic complementation analysis of cells with generalized peroxisomal dysfunction

Most patients with Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidemia are characterized by a deficiency of peroxisomes. We have developed a simple cytological method for the in situ detection of genetic complementation among and between these patients who are clinically and biochemically defined as having generalized peroxisomal dysfunction. This technique should facilitate both complementation studies in these disorders and investigations into the biogenesis of peroxisomes.

Spontaneous and 4-nitroquinoline 1-oxide-induced G2 chromosome aberrations in lymphoblasts from familial melanoma patients

Lymphoblastoid cell lines established from three unrelated kindreds with familial melanoma (FM) were cytogenetically analyzed for spontaneous and 4-nitroquinoline-1-oxide (4NQO)-induced chromosome aberrations. There were no significant differences between control, FM, or xeroderma pigmentosum (XP) cell lines for spontaneous aberrations. As a group, FM patients, as well as XP patients, had significantly higher 4NQO-induced aberrations than controls when metaphase cells were analyzed 2.5 hours after treatment during the G2 phase of the cell cycle. When selected cell lines were analyzed 18 hours after 4NQO treatment, the frequency of chromosome aberrations in FM cells returned to spontaneous levels, but XP cells retained significantly elevated aberration frequencies. The wide variability for chromosome aberrations within the control. FM relative, and FM patient groups during G2, however, indicated that analysis of total breakage rates alone would not be predictive of susceptibility to FM. Heterogeneity for carcinogen-induced chromosome breakage between some cancer-prone individuals and the possible significance of site-specific chromosome aberrations are discussed.

Fatty Acid Metabolism in Cultured Skin Fibroblasts from Patients with Peroxisomal Disorders: Lignoceroyl-CoA Ligase Deficiency in Childhood Adrenoleukodystrophy

We have previously reported that the peroxisomal B-oxidation system for very long chain (>C22) fatty acids is defective in the peroxisomal disorders, adrenoleukodystrophy (ALD) and Zellweger’s cerebro-hepato-renal syndrome (CHRS), (Singh, et. al. Proc. Natl. Acad. Sci. 81, 4203, 1984). In order to elucidate the specific enzyme defect, we examined the oxidation of [1-14C]lignoceric acid and [1-14C]lignoceroyl-CoA (substrates for the 1st and 2nd steps of the B-oxidation cycle). In agreement with our previous observation, we found that oxidation of lignoceric acid (substrate for the 1st step) in fibroblasts from childhood ALD was only 32% of the control. However, the rates of oxidation of lignoceroyl-CoA (substrate for the 2nd step) in cultured fibroblasts from childhood ALD and control were essential equivalent (44,853 ± 8,243 and 41,530 ± 3,708 CPM/mg protein/hr respectively). These studies indicate that oxidation of lignoceric acid is defective while degradation of lignoceroyl-CoA is normal in childhood AL. This identifies lignoceroyl-CoA ligase as the enzyme impaired in childhood ALD. Since lignoceric acid is oxidized in peroxisomes, it is likely that peroxisomal lignoceroyl- CoA ligase activity is defective in chilhood ALD. We also examined this oxidation in CHRS cells and found that degradation of both of these substrates is defective. These studies indicate that the molecular mechanism for the pathognomonic accumulation of very long chain fatty acids in X-linked childhood ALD is different from that in CHRS.

73 Rhizomelic Chondrodysplasia Punctata: Metabolic Studies in Isolated Peroxisomes

The Rhizomelic form of chondrodysplasia puntata (RCDP) is a fatal autosomal recessive peroxisomal disorders. Clinically, it is characterized by abnormal calcification of extremities, dwarfism, cataracts, skin charges and severe mental retardation. The biochemical findings were abnormal activities dihydroxyacetonephosphata acyltransferase (DHAP-AT) oxidation of phytanic acid whereas oxidation of lignoceric acid was normal.Peroxisomes isolated from two cell lines of RCDP and control each were compared for biochemical studies. The RCDP peroxisomes had the same density (1.178 gm/ml) as control peroxisomes. The residual activity (0.5% of control) of DHAP-AT was observed only in the peroxiscmes from RCDP and no such activity was observed in any other region of the gradient. The rates of activation and, oxidation of lignoceric acid was normal in peroxisomes from RCDP. The peroxisomes from RCDP contained 3-ketoacyl-CoA thiolase in the unprocessed form (44 KDa) whereas peroxisomes from normal peroxisomes contained both unprocessed (44KDa) and mature (41 KDa) forms. These results suggest that processing of 3-ketoacyl-CoA thiolase takes place in peroxisomes and recognition signals for its transport into peroxisomes were normal in RCDP.