Loss of the oncogenic phosphatase PRL-3 promotes a TNF-R1 feedback loop that mediates triple-negative breast cancer growth.

Stimulating tumor cell senescence and apoptosis are proven methods for therapeutically combating cancer. However, senescence and apoptosis are conventionally viewed as parallel, not sequential, processes. We have discovered that the metastasis-promoting phosphatase, PRL-3, is transcriptionally regulated by the NF-ĸB pathway in triple-negative breast cancer (TNBC) cells, and that PRL-3 knockdown elicits an autocrine tumor necrosis factor receptor 1 (TNF-R1) feedback loop that results in TNBC cell senescence followed by apoptosis. Knockdown of PRL-3 leads to rapid G1 cell cycle arrest and induction of a strong TNFα cytokine response that promotes a period of cellular senescence through TNF-R1-mediated activation of NF-ĸB. Senescent PRL-3 knockdown cells subsequently underwent apoptosis as a result of increased TNF-R1 signaling through the TNFα-associated extrinsic death pathway, shunting signaling away from the NF-ĸB cascade. These data suggest that TNF-R1 signaling dynamically re-programs after PRL-3 knockdown, from sustaining cell senescence through NF-ĸB to promoting apoptosis through TNF-R1 internalization and caspase-8 activation. The molecular mechanisms that determine the survival-death balance of TNF-R1 signaling are poorly understood, despite the fact that TNF-R1 has been extensively studied. Our results describe PRL-3 knockdown as a novel survival-death balance modifier of the TNF-R1 pathway, and show that senescent TNBC tumor cells can be sensitized to undergo apoptosis in a sequential manner.

31P-NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C- and 15N-NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states.

Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin-NADP+ reductase and, to a lesser degree, with those of NADPH-cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C'(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C'(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.

Expression and characterization of two pathogenic mutations in human electron transfer flavoprotein.

Defects in electron transfer flavoprotein (ETF) or its electron acceptor, electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II. In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired. Among these dehydrogenases are the four chain length-specific flavoprotein dehydrogenases of fatty acid beta-oxidation. In this investigation, two mutations in the alpha subunit that have been identified in patients were expressed in Escherichia coli. Of the two mutant alleles, alphaT266M and alphaG116R, the former is the most frequent mutation found in patients with ETF deficiency. The crystal structure of human ETF shows that alphaG116 lies in a hydrophobic pocket, under a contact residue of the alpha/beta subunit interface, and that the hydroxyl hydrogen of alphaT266 is hydrogen-bonded to N(5) of the FAD; the amide backbone hydrogen of alphaT266 is hydrogen-bonded to C(4)-O of the flavin prosthetic group (Roberts, D. L., Frerman, F. E. and Kim, J-J. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14355-14360). Stable expression of the alphaG116R ETF required coexpression of the chaperonins, GroEL and GroES. alphaG116R ETF folds into a conformation different from the wild type, and is catalytically inactive in crude extracts. It is unstable and could not be extensively purified. The alphaT266M ETF was purified and characterized after stabilization to proteolysis in crude extracts. Although the global structure of this mutant protein is unchanged, its flavin environment is altered as indicated by absorption and circular dichroism spectroscopy and the kinetics of flavin release from the oxidized and reduced protein. The loss of the hydrogen bond at N(5) of the flavin and the altered flavin binding increase the thermodynamic stability of the flavin semiquinone by 10-fold relative to the semiquinone of wild type ETF. The mutation has relatively little effect on the reductive half-reaction of ETF catalyzed by sarcosine and medium chain acyl-CoA dehydrogenases which reduce the flavin to the semiquinone. However, kcat/Km of ETF-QO in a coupled acyl-CoA:ubiquinone reductase assay with oxidized alphaT266M ETF as substrate is reduced 33-fold; this decrease is due in largest part to a decrease in the rate of disproportionation of the alphaT266M ETF semiquinone catalyzed by ETF-QO.

The mechanism for a 33-nucleotide insertion in mRNA causing sphingolipid activator protein (SAP-1)-deficient metachromatic leukodystrophy.

Metachromatic leukodystrophy is a severe autosomal recessive disorder caused by accumulation of sulfatide resulting from deficient lysosomal degradation. While most patients have mutations in the lysosomal enzyme arylsulfatase A, some patients have mutations in a required heat stable sphingolipid activator protein, we call SAP-1. One patient with SAP-1 deficiency was previously demonstrated to have a 33-nucleotide insertion in her mRNA. This resulted in the production of mature SAP-1 with 11 extra amino acids, which was unstable during intracellular processing. In this manuscript we demonstrate that the 33 nucleotides are present near the middle of a 4-kb intron, and that a single base change, c to a, in the second position preceding the 33-nucleotide insertion, coupled with the presence of a string of pyrimidines immediately upstream from this change, creates a new 3' splice junction. The presence of a string of pyrimidines within the 33-nucleotide insertion, which has three cag trinucleotides near the 3' end, leads to alternative splicing in normal people as found in this laboratory and by others. The insertion region is followed by a gt dinucleotide that is spliced to a typical 3' consensus sequence. The single nucleotide change, c to a, was confirmed by identifying normal and mutant sequence in the consanguineous parents and a sister, previously identified as a carrier of this disorder.

Insertion in the mRNA of a metachromatic leukodystrophy patient with sphingolipid activator protein-1 deficiency.

The lysosomal catabolism of sulfatide requires arylsulfatase A and a specific sphingolipid activator protein, SAP-1. While most patients with metachromatic leukodystrophy have mutations in the gene for arylsulfatase A, some patients have deficient SAP-1, as determined by immunological techniques. We now describe the molecular findings in a patient who died at 22 years of age with SAP-1 deficiency. The DNA polymerase chain reaction was used to amplify regions of cDNA which were subcloned in M13 phage DNA and sequenced by the dideoxy chain-termination method. The patient was found to have a 33-base-pair insertion between nucleotides 777 and 778 (numbered from the A of the ATG initiation codon). No other changes were found in the coding sequence of the cDNA from this patient. At the site of the insertion some normal people have an additional 9 base pairs, which correspond to the last 9 nucleotides at the 3' end of the insertion. The cDNAs from the second-cousin parents were amplified and sequenced, and in both two alleles were identified, one with the 33-base-pair insertion and one with no insertion. Two brothers were found to have only the normal alleles and a sister was found to have the 33-base-pair insertion and a normal allele. The findings confirm studies performed on leukocyte extracts demonstrating normal antigen levels in the two brothers and a lower level in the sister. The presence of 11 additional amino acids in the coding region of mature SAP-1 in this patient causes significant changes in the hydropathy profile compatible with the previous findings at the protein level.

Detection of a point mutation in sphingolipid activator protein-1 mRNA in patients with a variant form of metachromatic leukodystrophy.

The lysosomal degradation of sulfatide requires the specific enzyme, arylsulfatase A, as well as a heat stable protein called sphingolipid activator protein-1 (SAP-1). While most patients with metachromatic leukodystrophy have defects in arylsulfatase A, some patients have defects in SAP-1. SAP-1 is coded for by a gene on human chromosome 10 that also codes for three other proposed SAP. Examination of the cDNA from two siblings with SAP-1 deficiency revealed a point mutation of nucleotide #650 (counting from the initiation ATG) which is in the SAP-1 coding domain. This C to T transition changed the codon from threonine (ACC) to one coding for isoleucine (ATC). This eliminated the only glycosylation site in mature SAP-1 and could explain the findings made at the protein level.

Clinical, pathological, and biochemical studies on an infantile case of sulfatide/GM1 activator protein deficiency.

A 28-month-old black male died with severe complications of mental and motor deterioration, seizures, and aspiration. Autopsy demonstrated moderate liver enlargement, normal spleen and kidneys, small testes, and a grossly normal brain. Further examination showed irregular macrogyrae with evidence of a storage or sclerotic process. Thin layer chromatography of the lipids in formalin-fixed tissue demonstrated elevated levels of ceramide trihexoside and possibly sulfatides in liver and a decrease in the ratio of galactosylceramide to sulfatide in brain. Examination of the gangliosides in formalin-fixed brain indicated a slight increase in the percentage of GM1 ganglioside and a clear elevation in GM2 and GM3 gangliosides. Cultured skin fibroblasts had a normal activity for a large number of lysosomal enzymes including arylsulfatase A and galactocerebrosidase. When the cells were loaded with [14C]sulfatide only about 12% of the sulfatide was metabolized after 3 days. Extracts of the cells were subjected to SDS-PAGE and immunoblotting with antisphingolipid activator protein-1 (SAP-1) rabbit antiserum, and no cross-reacting material was detected confirming the diagnosis of metachromatic leukodystrophy caused by SAP-1 deficiency. This patient was clinically more severe than the other patients described previously with this deficiency. Further studies are underway to define the nature of the mutation in this patient.

Control of HL-60 cell differentiation lineage specificity, a late event occurring after precommitment.

Terminal cell differentiation of HL-60 promyelocytic leukemia cells results when they are continuously exposed to retinoic acid. This process involves an intermediate regulatory state, the precommitment memory state, which occurs before onset of differentiation or growth arrest in G0. The cellular processes occurring prior to onset of terminal differentiation can be resolved into early events anteceding development of the precommitment memory state and late events subsequent to it. While it has been suggested that retinoic acid induced early events regulate G1/0 specific growth arrest associated with terminal differentiation, the significance of induced late events is not known. Exploiting the capability of HL-60 cells to undergo either myeloid or monocytic differentiation in response to different inducers, the present studies examine the response of HL-60 cells to the sequential application of myeloid and monocytic inducers prior to onset of terminal differentiation. The results indicate that the precommitment state induced by retinoic acid is not differentiation lineage specific. Sequential application first of retinoic acid, a myeloid inducer, and then of 1,25-dihydroxyvitamin D3, a monocytic inducer, and vice versa, show that cellular choice of a specific differentiation lineage is regulated by late inducer driven events. The data support a two-step model for induction of terminal differentiation where early events anteceding precommitment regulate growth arrest and late events subsequent to precommitment regulate choice of a specific differentiation lineage. The results are of potential significance to the use of differentiation-inducing agents in chemotherapy. The potential toxicity of prolonged exposure to a single inducer might thus be mitigated by sequential brief exposures to different inducers.