Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1- to 2-μm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin.

The secreted mitogen vascular endothelial growth factor, VEGF, plays a pivotal role in angiogenesis. Hypoxia, inactivation of p53 and oncogenic K‐Ras induce VEGF expression. Other factors such as p73 may also affect VEGF levels. Curiously, p73 may also regulate angiogenesis by affecting the expression of the pigment epithelium‐derived factor, PEDF. Additionally, VEGF might harbor additional functions through the activation of E2F transcription factors. Recently, a new VEGF variant formed by alternative splicing, VEGF₁₆₅b, has been described as exerting anti‐angiogenic activity. We study here whether p73 isoforms levels ‐TAp73 and ΔTAp73‐ and p53 and K‐Ras status affect the expression of the above‐mentioned angiogenesis‐related genes (through the correlation between their expressions), the prognostic value of VEGF₁₆₅b and PEDF and the correlation between VEGF and E2F‐1 levels. Tumor and normal tissue of 112 colorectal cancer patients was analyzed to evaluate: (i) levels of TAp73, ΔTAp73, VEGF, VEGF₁₆₅b, PEDF and E2F‐1 by quantitative real‐time RT‐PCR, (ii) p53 status by immunohistochemistry and (iii) mutations in the first exon of K‐Ras by PCR‐SSCP. Tumor characteristics were examined in each patient. Associations were observed between: (i) specific p73 isoforms and VEGF and VEGF₁₆₅b expression; (ii) ΔEx2p73 variant and downregulation of PEDF; (iii) VEGF and PEDF expression; (iv) inactive p53 and VEGF₁₆₅b levels; (v) oncogenic K‐Ras and PEDF downregulation; (vi) E2F‐1 and VEGF expressions; (vii) VEGF₁₆₅b downregulation and poor prognosis parameters of tumors. We conclude that the levels of p73 isoforms could affect the expression of VEGF, VEGF₁₆₅b and PEDF. This scenario becomes complicated because a feedback between VEGF and PEDF may exist. VEGF may activate the E2F‐1 factor. Mutations in K‐Ras could negatively regulate PEDF expression. p53 inactivation might result in compensatory mechanisms such as over‐expression of VEGF₁₆₅b. Our data support the role of VEGF₁₆₅b as a tumor suppressor factor in colorectal carcinogenesis and its possible prognosis value.