Treatment of mammalian cells with 1,25-dihydroxyvitamin D3 (1,25D3) produces a G1 to S (G1/S) phase cell cycle block. In addition, it has been noted that a smaller proportion of cells accumulates in the G2/M compartment in 1,25D3-treated cultures. Since cyclins have a major influence on the regulation of cell cycle progression, we determined the expression of cyclins A and B as markers of the G2 phase and of cyclin E as the marker of G1/S transition. No increase in the steady-state levels of cyclin A or cyclin B mRNA was detected in the total cell population or in the cyclin B1 protein in the G2/M cell cycle compartment. In contrast, immunodetectable cyclin E protein was increased in cell cultures as a whole and specifically in the G2/M compartment cells. Determination of BrdU incorporation into DNA by flow cytometry showed marked inhibition of DNA replication in cells with DNA content higher than 4C, and autoradiography of 3H-TdR-pulsed cells showed that polynucleated cells did not replicate DNA after 96 h of treatment with 1,25D3 or analogs. Taken together, these experiments show that at least a portion of the G2/M compartment in 1,25D3-arrested cultures of HL60 cells represents G1 cells at a higher ploidy level, which are blocked from entering the high ploidy S phase.
Progression of mammalian cells through G1 is controlled by the concerted action of protein kinases, the activities of which are modulated in both positive (cyclins) and negative [cyclin-dependent kinase inhibitors (CDIs)] manners by families of regulatory proteins. In differentiation of leukemia cells, a G1 arrest is a common, if not invariable, occurence and takes place after the appearance of markers of monocytic differentiation in human leukemia HL60 cells treated with 1,25 dihydroxyvitamin D3 (1,25D3) at low to moderately high concentrations (F. Zhang et al., Cell Proliferation 27: 643-654, 1994). In the present study, we investigated the protein levels of several G1 regulatory proteins that are potential mediators of the 1,25D3-induced G1 block. During the first 24 h of exposure to a high concentration (4 x 10(-7) M) of 1,25D3, no increase was noted in the immunodetectable levels of cyclins D1 or E, or CDIs p16Ink4, p21Cip1/Waf1, or p27Kip1, even though monocytic differentiation markers were evident, and a prolongation of G1 was noted. After 48 h of exposure 4 x 10(-7) M to 1,25D3, a G1 to S-phase block progressively increased in parallel with the abundance of the p27Kip1 CDI. A transient increase in p21Cip1/Waf1 was noted only at 48 hr. The increase in p27Kip1 protein level was dependent on the concentration of 1,25D3 and was accompanied by an increase in cyclin D and E proteins, which normally peak in mid-G1 and at the G1 to S-phase transition, respectively. These results indicate that p27Kip1 protein is a strong candidate for the cell cycle regulator that blocks the entry into the S-phase in 1,25D3-treated HL60 cells.
The presentation of the Working Formulation for non-Hodgkin lymphomas by C. Berard was the highlight of 1-ICML, although at that time for Hodgkin lymphomas, the cell of origin was still unknown and even it could not yet be completely excluded, that Hodgkin lymphomas was not a neoplastic disease.Remembering that one realizes the huge evolution of our lymphoma understanding that has meanwhile taken place.ICML has hereby played a pivotal role not only as a platform for exchanging knowledge but also as an event catalyzing the initiation of collective efforts, which have led to some of the most significant achievements.So suffices here to mention the development of international prognostic index, the validation of the real classification, the definition of classification and staging of GI-non-Hodgkin lymphomas, the launch of international projects in the field of primary central nervous system lymphoma and peripheral T-cell lymphomas.This year was the third time that the closed workshop, which always proceeds the opening of ICML and which has been the starting point of the aforementioned projects, has been devoted to prognostic markers in diffuse large B-cell lymphoma under the heading 'Identification of diffuse large B-cell lymphoma subtypes: a way towards tailored treatment'.The formulation of the title of this year's workshop, the fact that the majority of the received abstracts for 12-ICML, are devoted to basic and translational research and the very structure of the current program, which is partly organized along pathways relevant both for the pathogenesis and the specific therapeutic targeting of lymphomas, underline the astonishing differences and the incredible progress realized between 1-ICML and 12-ICML.
We investigated the interfacial behavior of recombinant human cholesterol ester transfer protein (rCETP) using monolayer and surface balance techniques. rCETP bound to egg phosphatidylcholine monolayers spread at the air/water interface with a maximum surface pressure of 23 millinewtons (mN)/m at subphase concentrations between 3 and 5 x 10(-5) g/dl; the estimated dissociation constant was 7.5 x 10(-6) g/dl or 1 nM. The binding of rCETP to the lipid interface decreased linearly with increasing initial surface pressure; rCETP was excluded at pressures greater than 31 mN/m. rCETP catalyzed the desorption of [14C]cholesterol oleate from mixed lipid monolayers in a concentration dependent fashion. Similar studies with apolipoproteins A-I and A-IV established that cholesterol ester desorption was not caused by changes in surface pressure or cholesterol ester solubility. The desorption rate was proportional to subphase rCETP concentration, but at all concentrations surface radioactivity remained constant until surface pressure reached a plateau. The calculated binding stochiometry was one molecule of cholesterol ester desorbed for every 1000 molecules of rCETP in the subphase. We conclude that rCETP is surface active, binds to phospholipid monolayers with an affinity equivalent to that of the plasma apolipoproteins, and effects the desorption of cholesterol ester molecules from phospholipid monolayers by a carrier mechanism. Moreover, the relatively low equilibrium surface pressure of rCETP suggests that when bound to lipid the entire rCETP molecule may not penetrate the interface.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)