Increased stringency of the 1,25-dihydroxyvitamin D3-induced G1 to S phase block in polyploid HL60 cells.

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.

The effect of human cord blood on SJL/J mice after chemoablation and irradiation and its possible clinical significance.

There is evidence from the existing published literature that human umbilical cord blood, when used for purposes of bone marrow transplantation, does not necessarily have to be HLA matched in order to be efficacious. These reports include experimental observations on the ability of human umbilical cord blood to rescue lethally irradiated mice and clinical observations from China wherein HLA mismatched umbilical cord blood has been engrafted successfully in children with malignant disease. The study reported herein describes an experimental immunocompetent murine model to determine if human umbilical cord blood can be used to improve survival after chemoablation and irradiation. The animals received chemoablation followed by irradiation, and irradiation alone. The presence of human DNA in these mice following injection of human umbilical cord blood cells was determined, and the immunological status of the animals was evaluated. Animals receiving human umbilical cord blood cells after chemoablation and irradiation had a better mean survival at day 50 than animals receiving syngeneic marrow. Human DNA could be found in various organs, particularly the lung, spleen and liver of the mice for the first 30 days. Thereafter, human DNA became more difficult to detect but trace amounts of human DNA could be found up to one year later. The results of mixed lymphocyte reactions and phenotype analyses for murine T cell markers performed after injection of HUCB cells both indicated endogenous repopulation, and relatively intact immune systems in these mice. Since human umbilical cord blood allowed mice to survive the lethal effects of chemoablation plus irradiation, or irradiation alone, with reconstitution of the animals' own, relatively intact, immune systems, it would appear that HLA mismatched human umbilical cord blood could potentially be used as an adjuvant treatment for patients with advanced malignancies or other diseases for which hematopoietic reconstitution is indicated.

A dual block to cell cycle progression in HL60 cells exposed to analogues of vitamin D3.

The physiologically active form of vitamin D3, 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), induces differentiation of several types of myeloid leukaemia cells. The acquisition of monocyte-like phenotype is accompanied by slower progression through the cell cycle, and G1 block has been reported to be the basis of this effect. It is shown here that human promyelocytic leukaemia HL60 cells treated with analogues of vitamin D3 which are potent inducers of monocytic differentiation have an additional cell cycle block. Exposure to 10(-7) M 1,25(OH)2D3 or 1,25-(OH)2-16-ene-D3 resulted in monocytic differentiation and the expected G1 block evident at approximately 48 h in a rapidly differentiating variant of HL60 cells (HL60-G), and at 96 h in the more slowly differentiating HL60-240 cells. In addition, a G2+M block was noted at approximately 72 h in HL60-G and HL60-240 cells. Exposure to vitamin D3 analogues also markedly increased the number of dikaryons, suggesting that cytokinesis was impaired more than karyokinesis. Treatment with a third analogue 25-hydroxy-16,23-diene-D3 produced little differentiation and had minimal effects on the cell cycle parameters. These findings indicate that vitamin D3 analogues regulate cell proliferation by control of the transition of G1 and G2+M phases, reminiscent of the cdc2/CDK2 type of cell cycle control.

Demonstration of passenger leukocytes in a case of Epstein-Barr virus posttransplant lymphoproliferative disorder using restriction fragment length polymorphism analysis.

Lymphocytic populations of the posttransplant lymphoproliferative disorder in a completely matched renal allograft and a recipient's regional lymph node were examined, using restriction fragment length polymorphism analysis with probe YNH24 to the variable number tandem repeat D2S44. The donor's DNA was found in lymphocytes extracted from both the grafted kidney and the recipient's lymph node 26 days after engraftment. In both these organs, the results of in situ hybridization with a terminally biotin-labeled oligonucleotide probe to the Not I tandem repeat region of the Epstein-Barr virus (EBV) genome, as well as those of Southern blot hybridization to the EBV nuclear antigen region of the EBV genome, were positive. These findings confirmed the presence of the donor's lymphocytes ("passenger leukocytes") in the host nodal tissue in human renal transplantation and implicated EBV as playing a role in the development of posttransplant lymphoproliferative disorder. It is speculated that the EBV proliferative stimulus contributed to the recipient and the donor lymphocyte expansions. Alternatively, the proliferation of both lymphocyte populations could result from a mutual stimulation by minor histocompatibility or other antigens.

A new approach to the computer-assisted quantitative analysis of nuclear shape.

In recent years, researchers have explored the use of computer-assisted morphometric analysis to evaluate nuclear shape. Most of these studies use nuclear shape factors which are based on circumference/surface measurement ratios, such as variations of the nuclear contour index, the form factor, and the nuclear roundness factor. Here we present a new method for the objective evaluation of nuclear shape, involving a simple computer-assisted determination of nuclear area (N) divided by the area of a rectangular figure (F) with sides tangent to the nuclear margin and parallel to the frame of a video monitor. Following calculation of N/F for individual nuclei, our method generates statistical parameters for quantitating nuclear irregularity directly at the population level: the mean N/F ratio; standard deviation; and coefficient of variation. Our use of surface/surface measurement ratios makes our method independent of both magnification and nuclear size. Our method is applied first to normal lymphocytes and neutrophils to manifest the parameters for nuclear irregularity which are generated by our method. The sensitivity of our method is demonstrated using lymphoblasts from patients with acute lymphoblastic leukemia (ALL). Our objective ranking of nuclear irregularity for 20 cases of ALL correlates well with the subjective ranking of two pathologists. Because our method scores irregularity on a population basis and independently of other morphological criteria, it is compatible for use with the French-American-British (FAB) classification system (1981) for ALL.

Pink spots of Hedley-Whyte in formaldehyde-fixed brains. The subject revisited.

In an article published in 1985, Hedley-Whyte described "pink spots," which she observed in gross sections of human brains fixed in formaldehyde solution. Hedley-White found that the spots in question were associated with the presence of bacteria within central nervous system blood vessels. We have recently encountered three cases of Hedley-Whyte-type pink spots during routine postmortem brain examination. In two of these cases, the patient's clinical history included a bacterial infection, which could account for the deposition of bacteria within the central nervous system blood vessels. In the third case, the patient had no clinical history of bacterial infection, and had a negative postmortem blood culture. On microscopic examination, all three cases showed central nervous system intravascular presence of bacteria within the macroscopically recognized pink spots. To test the hypothesis that pink spots result from bacterial fermentation products, at the time of postmortem removal of brains (different from the brains mentioned above), we perfused blood vessels in three brains with 100% ethanol. Subsequent macroscopic examination after fixation in formaldehyde revealed discoloration similar to the Hedley-Whyte spots.