Cell kinetics of CD34-positive hematopoietic cells following chemotherapy plus colony-stimulating factors in advanced breast cancer.

Bone-marrow (BM) hematopoietic precursors are recruited into proliferative activity when colony-stimulating factors (CSF) are sequenced with chemotherapy (CT). Previous studies suggested that further CT can be safely administered only when the increased proliferative activity of these cells has subsided, because most cytostatic drugs selectively damage cycling cells. The safest interval between CSF discontinuation and the start of the next CT course needs to be ascertained in vivo. Thirty patients with advanced breast cancer were treated with an intensified FEC regimen, planned at 21-day intervals, sequenced with granulocyte-macrophage (GM)-CSF (15 patients) or granulocyte (G)-CSF (15 patients). Using flow cytometry (FCM) we evaluated the proliferation kinetics of CD34+ BM hematopoietic progenitors before CT+CSF and at different times after CSF administration was stopped. FEC+GM- and FEC+G-CSF sequences both induced a rapid and sustained increase in the percentage of BM myeloid precursors (BMMP%) and in the cycling status of CD34+BM cells. However, while the BMMP% remained elevated in both cases after CSF were stopped, the enhanced proliferative activity of CD34+ cells decreased more rapidly after GM- than after G-CSF. Using FCM, CD34+ BM-derived hematopoietic presursor cell kinetics is readily evaluated in the clinical setting. The administration of CSF following CT increases both the proliferative activity of CD34+ BM cells and the BMMP%. After CSF were discontinued a kinetic refractoriness of hematopoietic progenitors was more evident after GM-CSF than after G-CSF. These data may be of value in designing clinical trials to avoid cytostatic damage to the BM hematopoietic stem-cell compartment.

DNA content and cell kinetics in colorectal carcinoma: flow cytometric analysis of primary tumor and liver metastases.

AIMS AND BACKGROUND: Both flow cytometric DNA ploidy and proliferative activity have been indicated as potential prognostic indicators in colorectal cancer. Due to tumor biological heterogeneity, these parameters are best assessed with multiple sampling. METHODS: We undertook a prospective study on 52 patients with Duke's D colorectal tumors looking at multiple samples of the primary tumors and liver metastases. DNA ploidy and tumor proliferative activity (derived from proliferating cell nuclear antigen, PCNA FCM expression) were evaluated. RESULTS: Of primary tumors, 36/52 (69.2%) were aneuploid in at least 1 sample, with a median value of the DNA index of the aneuploid peak of 1.58. On liver metastases, 42/52 (80.7%) patients were aneuploid in at least 1 sample with a median DNA index of the aneuploid peak of 1.64. Identical or nearly identical histograms from different tumor samples were observed in only 18/52 (34.6%) of the primary tumors and in 15/52 (28.8%) of the liver metastases. The PCNA values for primary tumors ranged from 5 to 28% (median value = 16.5%). In the liver metastases, PCNA values ranged from 12 to 38% (median value = 19.8%). Proliferative activity was lower for diploid than for aneuploid tumors. DNA ploidy and PCNA expression of the deep specimen of primary tumors were similar to those of the liver metastasis of the same patient while this concordance was not complete in the case of superficial biopsy specimens. CONCLUSIONS: If correctly performed, FCM techniques allow an accurate analysis of DNA ploidy and proliferative activity and both these measurements can offer considerable potential for a more comprehensive approach to colorectal cancer.

In vivo enhanced antitumor activity of carmustine [N,N'-bis(2-chloroethyl)-N-nitrosourea] by simvastatin.

The effects of a combination of simvastatin, a cholesterol-lowering agent, and carmustine (BCNU; N,N'-bis(2-chloroethyl)-N-nitrosourea) on experimental C6 glioma were studied in vitro and in vivo. In vitro simvastatin and BCNU alone inhibited cell proliferation in a dose-dependent fashion. A subliminal concentration of simvastatin (0.1 microM) markedly and synergistically increased the BCNU toxicity to C6 glioma cells. The cytofluorimetric analysis of DNA from simvastatin-treated C6 glioma cells showed, besides the already described arrest in G1, an arrest/retardation in G2-M. Mitotic index from C6 cells incubated with simvastatin (10 microM) decreased by about 90%, indicating a specific C6 arrest/retardation in G2. The drug effects could be completely reversed by simvastatin withdrawal or mevalonate addition to the cultured cells. The combination of simvastatin and BCNU resulted predominantly from the profound retardation of cells in the G2-M compartment of the cell cycle. In vivo simvastatin (administered daily mixed with food) and BCNU (single i.p. injection), when given separately, caused a dose-dependent inhibition of labeling index in C6 glioma homografts (ID50, 61 mg/kg/day and 8.7 mg/kg, respectively). The combination of the lowest doses tested (simvastatin, 25 mg/kg/day and BCNU 0.3 mg/kg) resulted in a significant growth delay (compared to either drug alone) in C6 glioma (P < 0.05). There was no significant increase in toxicity as assessed by myelosuppression (WBC counts and bone marrow labeling index) and body weight. The results provide in vivo support for the combined use of simvastatin, a cholesterol-lowering agent, and BCNU in brain tumor treatment.

PKC activity in rat C6 glioma cells: changes associated with cell cycle and simvastatin treatment.

The parallel effects of simvastatin on cell cycle and PKC activity in rat C6 glioma cells were investigated. Simvastatin, 2.5 microM, for 24 h resulted in cell growth arrest in early G1 phase of the cell cycle and in a significant increase of total PKC activity (283 +/- 42 vs 470 +/- 61 pmoles/min/mg protein p = 0.002 for control cells and simvastatin-treated cells, respectively). The effect of simvastatin was fully prevented by mevalonate. A time dependent increase of PKC activity was observed in control exponentially free-growing C6 cells approaching confluency: a highly significant negative correlation (r = -0.91 p < 0.0001) between PKC activity and growth rate was calculated. PKC activity was high in cells arrested in G0 by serum starvation (0.4%). Following addition of complete medium (17.5% serum) the PKC activity progressively decreased and reached a minimum when cells traversed the G2/M phase, as determined by DNA analysis distribution. PKC activity dropped 30% in simvastatin-arrested early G1 cells; 44% in hydroxyurea-arrested cells at the G1/S boundary; and 73% in Colcemid mitosis-blocked cells. The results show that C6 glioma cell PKC activity is maximal in a G0 quiescent state and varies at different points of the cell cycle.

Oxidative lesion to membrane proteins in senescent erythrocytes.

The oxidative lesion undergone by membrane proteins in senescent human erythrocytes was evaluated by assaying their MetSO and thiol group content in ghosts and the amount of a coumarinyl derivative of maleimide, the DACM, bound by individual membrane proteins after treatment of erythrocytes of different age with this reagent. Quantitation of MetSO content of ghost membranes indicates an increase of the oxidative state of membrane proteins from young to mature and senescent erythrocytes, while thiol group assay does not show significant differences among erythrocytes of different age. Quantitation of DACM bound in intact cells by individual membrane proteins shows a decreased accessibility of thiol groups of band 3 protein and of the main proteins of the membrane skeleton in senescent erythrocytes, and this could be partly due to oxidation. The decreased reactivity to DACM of senescent erythrocyte band 3 seems to concern thiols located on the cytoplasmic domain of this protein, since the anion channel binds the same amount of the anion transport inhibitor EM, in mature and senescent erythrocytes.