Forced expression of the cyclin-dependent kinase inhibitor p16(INK4A) in leukemic U-937 cells reveals dissociation between cell cycle and differentiation.

OBJECTIVE: The aim of this study was to investigate how the tumor suppressor protein p16(INK4A) interferes with growth and differentiation of leukemic U-937 cells. MATERIALS AND METHODS: U-937 clones constantly overexpressing the cyclin-dependent kinase inhibitor p16(INK4A) were established. Clones transfected with empty vector were used as controls. The effects of high-level expression of p16(INK4A) on proliferation and cell cycle progression were investigated (cell cycle distribution, proliferation rate, analyses of different cell cycle regulatory proteins). The effect of introduction of p16(INK4A) on capacity for induced differentiation, assayed by capacity to reduce nitroblue tetrazolium, was determined. RESULTS: Overexpressed p16(INK4A) protein was active as judged by its ability to bind to CDK-4 in a coimmunoprecipitation assay. Clones overexpressing p16(INK4A) grew slower than controls, without any apparent effects on the phosphorylation status of the retinoblastoma protein (pRb). Instead, p16(INK4A) overexpression affected the phosphorylation status of pRb-related pocket protein p130, which was detected in its growth-restraining hypophosphorylated form. Despite an enhanced tendency to accumulate in G(0)/G(1), p16(INK4A)-overexpressing cells were less sensitive to induction of differentiation with vitamin D(3) or ATRA than control cells. CONCLUSIONS: Constitutive expression of p16(INK4A) in U-937 cells resulted in decreased proliferation as a result of activated p130 rather than pRb. Also, we showed that introduction of p16(INK4A) into U-937 cells impaired their capacity to differentiate. Moreover, the results support the notion that cell differentiation and cell cycle progression are dissociated and independently regulated processes.

Distinct requirements for optimal growth and In vitro expansion of human CD34(+)CD38(-) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells.

Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34(+)CD38(-) phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34(+)CD38(-) BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34(+)CD38(-) progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.

Sacrococcygeal teratoma in Sweden: a 10-year national retrospective study.

Thirty-two children with sacrococcygeal teratoma have been treated during the last 10 years (1980 to 1989) in Sweden. A retrospective study was performed in four departments of pediatric surgery that treat sacrococcygeal teratomas in children from the whole of Sweden. Prenatal and perinatal histories were reviewed together with interval to diagnosis, Altman classification, histology, and serum alpha-fetoprotein. Details of surgical management +/- adjuvant chemotherapy and outcome of patients were also documented. In 8 patients the teratoma was diagnosed prenatally by ultrasonography and there was one postoperative death in this group. Multiagent chemotherapy was used in all but one of 11 patients with malignant teratomas (in 8 of them a cisplatin, bleomycin, vinblastine combination). Only one patient with a malignant tumor treated by single-agent chemotherapy died, 8 others were still alive and tumor-free after 1 to 9 years (mean time, 5.4 years). Two patients developed late relapses and were treated by surgical resection. Metastases occurred in five of the 11 malignant tumors, one at presentation and in four patients 10 to 29 months following surgery. All relapses had distant metastases as well as local disease. Serum alpha-fetoprotein was used in monitoring some of these patients.

Relation of length of submucous segment of the ureter to lesions of the renal parenchyma in children.

The possible relationship between the length of the submucous ureteral segment and frequency and degree of renal parenchymal injury was analysed in children with urinary tract infection and vesico-ureteral reflux. If the grade of reflux was disregarded, no relation was found to exist between these parameters.

Oesophageal clamp anastomosis and stricture formation. An experimental study in the piglet.

End-to-end oesophageal anastomoses were performed with a metal clamp in 8 piglets. A transanastomotic catheter was kept in position for 5 weeks following release of the clamp. The results were evaluated by contrast radiography after a further 2 weeks. All the animals survived the operation and the pre-determined observation period. All the anastomoses healed without signs of leakage. They showed strictures of varying degrees, as assessed by radiography of the specimens. The dissected specimens showed scarred changes in the anastomoses where the thin mucosa was regenerated. The results are explained by the fact that the method of clamp anastomosis per se leads to a mucosal defect at the site of anastomosis with ensuing secondary wound healing. The intraluminal tube therby prevents wound contraction with luminal obliteration by giving the mucosa time to regenerate.