Differential transactivation by alternative EWS-FLI1 fusion proteins correlates with clinical heterogeneity in Ewing's sarcoma.

The t(11;22)(q24;q12) translocation is present in up to 95% of cases of Ewing's sarcoma and results in the formation of an EWS-FLI1 fusion gene which encodes a chimeric transcription factor. The proximate role of EWS-FLI1 in the pathogenesis of Ewing's sarcoma is thought to involve the activation of as yet largely unknown target genes. Many alternative forms of EWS-FLI1 exist because of variations in the locations of the EWS and FLI1 genomic breakpoints. The most common form, designated "type 1," consists of the first seven exons of EWS joined to exons 6-9 of FLI1 and accounts for approximately 60% of cases. The "type 2" EWS-FLI1 fusion also includes FLI1 exon 5 and is present in another 25%. We and others have observed previously that the type 1 fusion is associated with a significantly better prognosis than the other fusion types. Because EWS-FLI1 is an aberrant transcription factor, we investigated whether these differences in clinical behavior may be correlated to functional differences by comparing transactivation by the type 1 EWS-FLI1 with other types in both heterologous cells (HeLa, NIH3T3) and homologous cells (Ewing's sarcoma cell lines). In a panel of seven Ewing's sarcoma cell lines, we found transactivation of a transiently transfected FLI1-responsive reporter construct to be significantly lower in cell lines with the type 1 fusion than in cell lines with the type 2 fusion (P = 0.003). Cotransfection of the same reporter construct with each of a series of seven EWS-FLI1 expression constructs (corresponding to the two major fusion types and five less common types) also showed that type 1 EWS-FLI1 was a significantly weaker transactivator than the type 2 product in both HeLa and NIH3T3 cells (P = 0.003, and P = 0.033, respectively). Electromobility shift assays showed equivalent binding of the type 1 and type 2 EWS-FLI1 to the consensus FLI1-responsive binding site, indicating that differences in transactivation were not due simply to differences in DNA binding affinity. The finding that the type 1 EWS-FLI1 fusion, associated with less aggressive clinical behavior, encodes a less active chimeric transcription factor may provide the basis for a molecular explanation of clinical heterogeneity in Ewing's sarcoma.

Acyl-coenzyme A: cholesterol acyltransferase and cholesterol ester hydrolase in the outer and inner cortices of the guinea pig adrenal: effects of adrenocorticotropin and dexamethasone.

The guinea pig adrenal cortex can be separated into two zones with contrasting lipid contents: a lipid-rich outer cortex and a lipid-poor inner cortex. Cytoplasmic lipid droplets contain cholesterol esters and are thought to act as a reservoir for steroid precursor. Such lipid droplets are numerous in the outer cortex, but accumulate in the inner region only after treatment with ACTH. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) esterifies cholesterol to fatty acids for storage in this pool, while cholesterol ester hydrolase (CEH) cleaves these esters, increasing free cholesterol. However, the relationship between intracellular cholesterol content and the activity of these enzymes is not clear. In this study we examine the capacity to esterify cholesterol (ACAT) and to hydrolyze cholesterol esters (CEH) in the lipid-rich and lipid-poor regions of the guinea pig adrenal in control animals and animals treated with ACTH or dexamethasone (DEX). For all conditions, the lipid-filled outer cortex possessed more ACAT and CEH activity than did the inner. Although ACAT and CEH in the outer cortex were relatively insensitive to ACTH compared to the enzymes in the inner zone, they were significantly suppressed after DEX treatment. This implies that in the basal state, ACAT and CEH may be almost maximally stimulated in the outer cortex, and that in these cells, cholesterol esterification and hydrolysis may occur at a high rate even in the basal state. The inner cortex responded to ACTH with increased ACAT activity, but was little affected by DEX. In the inner zone, CEH activity was not affected by ACTH or DEX. The lower level of these enzymes in the inner cortex correlates with the paucity of lipid droplets in these cells in the basal state, while the increase in ACAT, but not CEH, in this zone after ACTH treatment correlates with the accumulation of small lipid droplets in inner cortical cells of ACTH-treated animals.

3-Hydroxy-3-methylglutaryl coenzyme A reductase in outer versus inner cortices of the guinea pig adrenal: effects of adrenocorticotropin and dexamethasone.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, a rate-limiting enzyme in cholesterol biosynthesis, was examined in the lipid-filled outer cortical cells and smooth endoplasmic reticulum-filled inner cortical cells of guinea pig adrenals. The specific activity of HMG CoA reductase was higher in microsomes obtained from the outer cortex, but was stimulated by ACTH and suppressed by dexamethasone (DEX) in both regions. Immunoblots of the microsomal proteins revealed a higher concentration of immunoreactive HMG CoA reductase (mol wt 94K) in microsomes from outer cortical cells. Changes in the intensity of this band corresponded to changes in activity after treatment with ACTH and DEX. However, quantitative immunoassay revealed that changes in activity in the inner zone after ACTH and in both zones after DEX were greater than could be accounted for by changes in immunodetectable protein, implying that other regulatory factors play a role in these cases. Incubation of outer and inner cortical tissues in vitro showed that outer cortical tissue had a greater capacity to incorporate [14C]acetate into cellular cholesterol (free and esterified) and into secreted steroid than did inner cortical tissue. ACTH enhanced the incorporation of [14C]acetate by outer cortical tissues into secreted steroid, but had little effect on incorporation by inner cortical tissues. Assay of the medium indicated that outer cortical tissues secreted much more steroid and increased secretion in response to ACTH, whereas inner cortical tissues secreted little steroid and were little affected by ACTH. Taken together, these results show that outer cortical cells have a greater capacity for cholesterol synthesis and that enhancement of this capacity after ACTH treatment is correlated with an increase in HMG CoA reductase protein. On the other hand, while the level of HMG CoA reductase immunodetectable protein and activity in inner cortical cell microsomes is considerable and appears to increase after ACTH treatment, it is not translated into an ability to synthesize cholesterol. This suggests that the activity of the immunodetectable HMG CoA reductase in the inner zone is suppressed in vivo and that the prominent smooth endoplasmic reticulum found in inner cortical cells has additional functions. The inability of the inner zone to synthesize cholesterol accounts in part for its low steroid production.

Immunohistochemical detection of hepatitis C antigen by monoclonal antibody TORDJI-22 compared with PCR viral detection.

We sought to determine the sensitivity and specificity of immunohistochemistry using the TORDJI-22 MoAb (BioGenex, San Ramon, Calif), which is specific for the C-100 protein of the hepatitis C virus, compared with reverse transcriptase-polymerase chain reaction (RT-PCR) of tissue for viral RNA. RT-PCR had been performed on 52 fixed tissue specimens. Immunohistochemistry was performed using prediluted antibody with the alkaline phosphatase/fast red (BioGenex) technique. Predigestion with Protease XXIV (BioGenex) and other procedures followed the manufacturer's protocols. Positive immunohistochemistry was narrowly defined as tightly clumped, perinuclear red granules in hepatocytes. Of the specimens, 28 were positive by RT-PCR. With RT-PCR as the standard of comparison, immunohistochemistry yielded a sensitivity of 70% and specificity of 84%. Positive cells, when present, were usually very rare. With stringent criteria, immunohistochemistry with the TORDJI-22 monoclonal antibody is a very specific, fairly sensitive diagnostic test for hepatitis C virus in fixed liver tissues.

Immunocytochemical characterization of intrarenal adrenal tissue.

Heterotopic adrenal tissue has been reported in multiple sites, but its functionality has seldom been assessed. In this case, immunocytochemistry was used to characterize adrenocortical tissue present in the subcapsular region of a nephrectomy specimen and to determine its potential for steroidogenesis. Immunodetectable cytochrome P450scc was detectable in the adrenal gland, but not in the renal tissue, clarifying the demarcation between the two tissue types. The high level of this key enzyme in steroid synthesis in the adrenocortical cells suggested that they were capable of producing steroids.

beta-Carotene transport in human lipoproteins. Comparisons with a-tocopherol.

The purpose of this study was to investigate the temporal relationships of the transport of beta-carotene in human lipoproteins. We administered 60 mg beta-carotene with breakfast to nine fasting subjects, then blood samples were collected at intervals of up to 75 h, lipoproteins were isolated, and beta-carotene was quantitated. beta-Carotene concentrations in chylomicrons and very low density lipoproteins (VLDL) peaked at 6 and 9 h, respectively. Nonetheless, at all time points the majority of plasma beta-carotene was contained in low density lipoproteins (LDL), while high density lipoproteins (HDL) carried a smaller portion (at 24 h, 73 +/- 8% in LDL as compared with 23 +/- 5% in HDL). In three subjects, transport of beta-carotene was compared with the results of earlier studies on the transport of stereoisomers of alpha-tocopherol. Unlike plasma RRR-alpha-tocopherol concentrations, which are maintained by the preferential incorporation of RRR-alpha-tocopherol into VLDL by the liver, beta-carotene increased and decreased in VLDL similarly to SRR-alpha-tocopherol, a stereoisomer whose concentrations are not maintained in plasma. In conclusion, beta-carotene is primarily transported in the plasma in LDL, but its incorporation by the liver into lipoproteins does not appear to be enhanced.

Differential ACTH response of immunodetectable HMG CoA reductase and cytochromes P450(17 alpha) and P450(21) in guinea pig adrenal outer zone cell types, zona glomerulosa and zona fasciculata.

In the guinea pig adrenal the cells of the outer zone secrete high levels of steroid and respond to ACTH with increased synthesis of cholesterol and steroid. The outer zone consists of two cell types: zona fasciculata (ZF) and zona glomerulosa (ZG). To determine the relative contribution of ZF and ZG to the outer zone's ACTH response, purified populations of each cell type were prepared from ACTH-treated and control animals. Levels of proteins potentially involved in the ACTH response were measured by ELISA. HMG CoA reductase, the rate limiting enzyme of cholesterol synthesis, and two cytochrome P450s, P450(17 alpha) and P450(21), were studied. P450(17 alpha) is required for production of cortisol, but not for corticosterone or aldosterone. P450(21) is required for production of all of these corticosteroids. ZF cells had 4-5 fold greater concentration of all three proteins, but the proteins in ZG cells showed a greater response to ACTH (approximately 3 fold). The response of ZG cells to ACTH and their possession of P450(17 alpha) is consistent with observations made in vitro that ZG cell populations synthesize cortisol and respond to ACTH with increased output of cortisol as well as of corticosterone and aldosterone. In ZG cells to a greater extent than ZF cells, the response to ACTH involved increases in levels of enzymes of both cholesterol and steroid synthesis, suggesting that new protein synthesis is an important component of their ACTH response. On the other hand, the fact that ZF cells can increase steroid output in response to ACTH with a lesser increase in these proteins suggests a different mechanism of regulation. Mobilization of stored cholesterol may be more important in the ACTH-responsiveness of the lipid-filled ZF cells than in the lipid-poor ZG cells.

Immune complex glomerulonephritis in patients coinfected with human immunodeficiency virus and hepatitis C virus.

Human immunodeficiency virus-associated nephropathy (HIVAN), characterized by heavy proteinuria, rapidly progressive renal failure, "collapsing" glomerulopathy, and tubulointerstitial abnormalities, is the most common finding in HIV-infected patients undergoing a renal biopsy and predominantly affects blacks. We describe the clinical features and renal pathologic findings of 12 intravenous drug users (IVDUs) coinfected with HIV and hepatitis C virus (HCV) who were selected for renal biopsy because they presented with features different from typical HIVAN, including hypertension, microscopic hematuria, and cryoglobulinemia. There were seven black and five Hispanic patients. Eleven patients had immune complex glomerulonephritis (ICGN); one had glomerulosclerosis with immune complex deposits. Ten individuals had evidence of past hepatitis B viral infection, but none had persistent hepatitis B surface antigenemia. No other underlying cause for immune complex glomerulonephritis was identified. Renal biopsy showed membranoproliferative glomerulonephritis in five patients, mesangial proliferative glomerulonephritis in five, membranous nephropathy in one, and "collapsing" glomerulopathy with immune complex deposits in one. Hepatitis C virus RNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in the renal tissue and/or serum of nine of the 11 patients tested, and also in the renal biopsy tissue of four of eight patients with clinical and pathologic features of typical HIVAN without immunofluorescence evidence of immune complex deposits. One patient presented with renal failure, five patients developed end-stage renal disease (ESRD) requiring hemodialysis (mean time, 6.5 months), and six had stable renal function after a mean follow-up of 29.1 months (range, 2 to 72 months). Liver function abnormalities were present in seven of the 12 individuals, including four of the six patients who developed renal failure. These findings indicate that in some patients coinfected with HIV and HCV, the development of ICGN may dominate the clinical course of the disease. The occurrence of ICGN among black patients at risk for HIVAN may be related to the relatively high prevalence of HCV infection among IVDUs in this group.

Molecular analysis of the fusion of EWS to an orphan nuclear receptor gene in extraskeletal myxoid chondrosarcoma.

The pathogenesis of myxoid chondrosarcoma (CS) is poorly understood. A recurrent translocation, t(9;22) (q22;q12), has been recognized in CS, specifically in extraskeletal myxoid CS. Recently, this translocation has been shown to represent a rearrangement of the EWS gene at 22q12 with a novel gene at 9q22 designated CHN (or TEC). Sequence analysis suggests that CHN encodes a novel orphan nuclear receptor with a zinc finger DNA-binding domain. The structure of this gene fusion has been characterized in only a limited number of extraskeletal myxoid CSs and its presence in other types of CS has not been extensively examined. We studied 46 cases of CS (8 extraskeletal myxoid, 4 skeletal myxoid, 4 mesenchymal, and 30 other) for the EWS/CHN gene fusion by reverse transcriptase polymerase chain reaction, Southern blotting, and long-range DNA polymerase chain reaction. The EWS/CHN gene fusion was present in 6 of 8 extraskeletal myxoid CSs and was not detected in any of the remaining cases, including the 4 skeletal myxoid CSs. The negative findings in the latter cases suggest that skeletal myxoid CS is pathogenetically distinct from its extraskeletal counterpart. Notably, 2 cases of extraskeletal myxoid CS showed neither an EWS/CHN fusion transcript nor EWS/CHN genomic fusion nor EWS or CHN genomic rearrangement, suggesting genetic heterogeneity within extraskeletal myxoid CS. Finally, we also provide evidence for alternative splicing of the 3' end of the fusion transcript. Extraskeletal myxoid CS thus represents yet another sarcoma type containing a gene fusion involving EWS.