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Introduction: Nowadays UCB represents an established source of hematopoietic stem cells for unrelated transplants in children and the employ in adults is quickly growing up. Nucleated cells (NCs) content is one of the main predictors to evaluate UCB for clinical use; however, other indicators of the hematopoietic potential, such as CD34+ cell and colony-forming cells (CFUs), have recently showed similar correlations. According to Netcord-FACT standards, it is recommended to test all the above mentioned parameters before releasing UCB to the transplant center; a segmented tubing of the UCB bag should be used but a satellite cryotube is more often available. We preliminarily report the results of quality controls performed on thawed cryovials corresponding to each of 15 units delivered by our UCB Bank. Material and methods: in our policy, all UCBs are stored accompanied by 3 satellite cryovials, treated under the same conditions of the unit. For each of the 15 UCBs released for transplantation, one cryotube was thawed in a 37°C water bath with a gentle agitation, without washing out DMSO. NCs and mononucleated cells (MNCs) were estimated with an automated cell counter. Viability and CD34+ cell count were evaluated by flow cytometry, with a no-wash, single-platform technique and 7-aminoactinomycin D. CFU assay was performed using commercial reagents (Methocult GF H4434, StemCell Technologies) and colonies were classified after a 14 days incubation. The same parameters referring to fresh UCBs (before cryopreservation) were always available. Results: the UCB characteristics measured after thawing a cryovial were compared with those of the fresh cord and are detailed in the table below. fresh UCB before cryopreservation UCB cryovial after thawing Yield (%) NC (x106) 1491.3 (148–2262) 1354.2 (167.4–2119.9) 92 (83–113) MNC (x106) 662.7 (96.5–900.3) 638.8 (295–1238.7) 95 (63–159) CD 34+ cells (x106) 3.47 (0.38–10.87) 3.19 (0.4–9.27) 85 (37–118) Viability (%) 96 (88–100) 62 (39–77) Viability of CD34+ cells (%) 92 (71–99) CFU-GM (x104) 838.5 (57.2–3581.2) 424.11 (65.29–917.64) 69 (16–184) BFU-E (x104) 1709.61 (159.84–5116) 473.34 (39.69–1204.14) 41 (5–139) total CFU (x104) 2565.17 (217.04–8953) 911.68 (164.43–2075.22) 57 (9–160) Excellent yields were found for NCs, MNCs and CD34+ cells. Despite of the decrease in the overall viability, the viable CD34+ cells as percentage was always highly satisfactory. The colonies growth was found lower in the thawed sample in comparison with fresh UCB before cryopreservation. Conclusion: in our experience, highly satisfactory evaluations of UCB content could be obtained using thawed cryotubes with regard to NC, MNC and also CD34+ cell. However, concerning the latter, the different methods employed on fresh UCBs, such as CD34+ cells detection without beads, may be advocated to explain some discrepancies in the yield range. The results of CFU assay confirmed to be poorly useful, essentially because affected by a subjective interpretation even if the reduced cell growth may be also related to the presence of DMSO as inhibiting factor.

The human ARF/INK4a locus encodes two cell cycle inhibitors, p16(INK4a) and p14(ARF), by using separate promoters. A variety of mitogenic stimuli upregulate ARF but a direct modulation at the transcriptional level has been reported only for E2F-1. We show here that the ARF promoter is strongly responsive also to E2F2 and E2F3, thus providing a strong support to their suggested role in the induction of apoptosis. Through the usage of both deletion mutants and/or site-directed mutants, we surprisingly found that none of the four putative E2F consensus sites is strictly necessary for the upregulation of ARF expression, as a minimal deletion mutant, lacking all the putative E2F binding sites, is still transactivated by E2F. Moreover, our data suggest that the ARF promoter is regulated by E2F through both direct binding to the promoter sequences and indirectly, probably by being tethered to the ARF promoter by Sp1-like factors.

Genetic alteration of the p53 tumor suppressor gene, which monitors DNA damage and operates cell cycle checkpoints, is a major factor in the development of human malignancies. The p53 protein belongs to a family that also includes two structurally related proteins, p63 and p73. Although all three proteins share similar transcriptional functions and antiproliferative effects, each of them appears to play a distinct role in development and tumor suppression. One of the principal regulators of p53 activity is the MDM2 protein. The interaction of MDM2 with p53 inhibits p53 transcriptional activity and targets p53 for ubiquitin-dependent degradation. The ability of MDM2 to inhibit p53 functions is antagonized by the ARF oncosuppressor protein. We show here that like p53, the p63alpha and p63gamma isoforms are able to associate with human MDM2 (HDM2). Overexpression of HDM2 increased the steady-state level of intracellular p63 and enhanced its transcriptional activity. Both effects appeared to be counteracted by ARF coexpression. These data indicate that p63 can be activated by HDM2 under conditions in which p53 is inhibited. Therefore, HDM2 expression could support p63-specific transcriptional functions on a common set of genes, keeping interference by p53 at a minimum.

The INK4a gene, one of the most frequently disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p19ARF, that both block cell proliferation. p16INK4a is a component of the Rb regulatory pathway, while p19ARF has been functionally related to p53. Moreover, p16INK4a is inactivated in many human tumors, while it has been very recently reported that p19ARF null mice develop tumors early in life. We show here that p19ARF is able to inhibit the formation of G418-resistant colonies when transfected into human and mouse cell lines expressing wild-type p53, regardless of p16 status. Moreover its amino terminal domain encoded by exon 1beta is still sufficient to obtain the same effect. We have analysed the ability of p19ARF to interfere with Ras-mediated cellular transformation in the NIH3T3 cell line. Cotransfection of p19ARF together with activated ras potently inhibited the formation of transformed foci in a dose-dependent manner. We have also isolated stable NIH3T3 transfectants expressing p19ARF and we have measured their growth properties as well as their efficiency of transformation by activated ras. Our results suggest that p19ARF can interfere with oncogene-mediated transformation, without significantly affecting NIH3T3 cell growth, at least at the levels of expression achieved in these experiments.

The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16(INK4a) and p14(ARF) (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.