Isolation and characterization of an RNA-proteolipid complex associated with the malignant state in humans.

Abstract

An RNA-proteolipid complex was isolated from sera of patients with a variety of malignant disorders as well as from culture media of malignant cell lines. The complex, characterized by a relatively constant composition, contains 27S poly(A)+ RNA and Mr 1250 oligopeptide(s) and is rich in phospholipids and glycosphingolipids. Serum (Behring, Marburg, FRG). For fractionation of the RNA-proteolipid complex into components, aliquots (1 ml) of each fraction were further dialyzed in benzoylcellulose bags against distilled water and lyophilized.Gross Separation of the Proteolipid From RNA. The Iyophilized samples were suspended in 5 ml of chloroform/methanol (2:1, vol/vol) and stirred for 3 hr at 220C. Centrifugation at 10,000 x g for 10 min at 40C yielded a pellet which was reextracted with 2.5 ml of chloroform/methanol (1:1.5, vol/ vol). The organic-soluble phases were pooled and evaporated to dryness. The organic-soluble fraction (designated fraction 1), which was free of RNA, was further fractionated to examine its peptide and lipid content. The precipitate, fraction 2, was rich in RNA and was independently fractionated.Fractionation ofpeptides and lipids infraction 1. Fraction 1 was solubilized in 2 ml of chloroform/methanol (2:1); it then was added to 3 volumes of diethyl ether and kept at 40C for 10 hr to precipitate a peptide-rich fraction, which was collected by centrifugation at 10,000 x g for 10 min at 40C. The supernatant (Li) was analyzed for lipid composition directly (see below). Since the peptide-rich fraction would readily not dissociate from firmly bound glycosphingolipids and phospholipids, it was extracted with 2 ml of chloroform/ methanol (1:1) acidified to pH 2 with HCl. The extraction was repeated twice, yielding a peptide precipitate (P1) and a pooled lipid extract (L2). The delipidated oligopeptides were collected by centrifugation and dissolved in distilled water for estimation and further characterization.Fractionation of RNA-rich fraction 2. The insoluble fraction was extracted twice with 2 ml of 65% ethanol at 22°C for 2 hr. The soluble material was dried in vacuo and then was extracted twice with acidified chloroform/methanol (1:1) to obtain a peptide fraction (P2) and the soluble lipid fraction (L3). The ethanol-extracted pellet was dissolved in 100 mM NaCl, and RNA was further purified from peptides by three cycles of precipitation at -70°C with 2 volumes of ethanol. The pooled supernatants yielded another peptide fraction (P3).Lipid Analysis. Fractions L1-L3 were pooled and analyzed on silica H gel (Merck), together with standard lipids (Sigma and Seromed, Munich, FRG), in chloroform/methanol/H20 (60:35:8, vol/vol) and hexane/diethyl ether (70:30, vol/vol). Cholesterol was analyzed by gas chromatography with stigmasterol as a standard. Phospholipids isolated by chromatography were determined by the method of Bartlett (5). Glycosphingolipids (ceramide hexasaccharides) were determined by gas chromatography of the trimethylsilylated methyl glycosides as described (6).Peptide An...

Real-Time Quantification in Plasma of Human Telomerase Reverse Transcriptase (hTERT) mRNA: A Simple Blood Test to Monitor Disease in Cancer Patients

“…Our preliminary results show that real-time measurement of hTERT mRNA in plasma discriminates between healthy subjects and cancer patients suffering from colorectal cancer or follicular lymphoma and support the idea that a diagnostic or prognostic test for cancer might be developed based on this genetic marker in blood. This observation agrees with previous studies suggesting that RNA might be present in plasma or serum (Wieczorek et al, 1985(Wieczorek et al, , 1987 and with more recent reports that human mRNA of tyrosinase (Kopreski et al, 1999) and telomerase (Chen et al, 2000) can be extracted and amplified from the serum of patients with malignant melanoma and breast cancer, respectively. Here, we have shown that real-time PCR of hTERT could be used in the future and might increase the number of cases detected by revealing the number of copies in healthy persons and in cancer patients, particularly when considering that the presence in plasma of tumor-derived mRNA is not limited to any given cancer type but may be a relatively frequent finding in a wide range of cancers.…”

Relevance of circulating tumor cells, extracellular nucleic acids, and exosomes in breast cancer

“…Questions remain as to the origin of extracellular nucleic acids, which can be found as free nucleic acids [67], bound to a cell's surface via proteins with specialized nucleic acid binding properties [68][69][70], complexed with proteolipids [71,72] and within apoptotic bodies [73]. Studies have shown that they (DNA and RNA) can be released spontaneously from cells [74,75].…”

Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells

“…The second is a phospholipid metabolically related to the former. These findings are discussed in view of the analogies with the complexes found in the sera [2][3][4][5] and those isolated from the culture media of cells [1].…”

“…The well-assessed phenomenon of vesicle shedding from tumor cells [1] deserves much attention mainly in relation to the observation that similar membrane vesicles [2] or RNA-proteolipid complexes [3][4][5] circulate in the blood of patients with cancer. Our previous work [6][7][8] has demonstrated that RNA is present in vesicles released from the Abbreviations."…”