Growth-inhibitory effects of coumarin (1,2-benzopyrone) and 7-hydroxycoumarin on human malignant cell lines in vitro.

Coumarin (1,2-benzopyrone) is a natural substance that has shown antitumor activity in vivo. The major human metabolite of coumarin, 7-hydroxycoumarin (7-HC), is the active form of the drug. While the exact mechanism(s) of action of coumarin is unknown, it has been shown previously that this drug possesses immunomodulatory activity in vitro and in vivo. The present investigations examined the direct (non-immunological) antitumor effects of coumarin and 7-HC in vitro. Both coumarin and 7-HC were found to be growth-inhibitory (cytostatic) for the following human malignant cell lines: A549, ACHN, Caki-2, Dakiki, HS-Sultan, H727, HCT-15, HL-60, K562, LNCaP, PC-3, Du 145 COLO-232, MCF-7 and RP-1788. The growth inhibition was dependent on dose and time and was reversible upon removal of cells from medium containing the drug. Coumarin and 7-HC inhibited [3H]thymidine, [3H]uridine and [3H]leucine incorporation. In a similar fashion, coumarin and 7-HC inhibited the intracellular production of prostate-specific antigen by LNCaP cells. Coumarin and 7-HC stimulated apoptosis in HL-60 cells but not in other cell lines tested. It is concluded that coumarin and 7-HC have direct antitumor (cytostatic) activity as well as immunomodulatory activity. Further information is needed in order to determine which activities are responsible for antitumor activity in vivo.

Combination of classical and interphase cytogenetics to investigate the biology of myeloid disorders: detection of masked monosomy 7 in AML.

Numerical abnormalities of chromosome 7 were detected by using fluorescence labeled in situ hybridization (FISH) procedure with a centromere-specific probe in four cases of acute myeloid leukemia (AML) and three cases of myelodysplastic syndromes (MDS). Comparison of these results with classical cytogenetic (CC) data demonstrated a good correlation between the two methods. FISH confirmed the finding of monosomy 7 in all patients who demonstrated this abnormality by CC. Two AML patients who did not show monosomy 7 by CC were unexpectedly found to contain this abnormality in 39.8% and 17% cells when examined by FISH. Given that our modified FISH method consistently yielded > 96% hybridization efficiency, these findings constitute an unexpected but real presence of monosomy 7 in a substantial number of interphase cells that had remained undetected by classical karyotyping. Finally, a number of maturing myeloid cells including granulocytes also demonstrated monosomy 7 by FISH, thereby confirming the ability of malignant cells to undergo differentiation. We conclude that FISH constitutes a highly sophisticated molecular technique which can be extremely useful in select cases for detecting 'masked monosomy 7' as well as helping to determine the lineage of terminally mature cells in AML, thereby providing a handle on the effects of cytokines or chemotherapy on normal vs leukemic clones.

A novel triple label method validates the double label technique of defining cell cycle kinetics in HL-60 cells.

HL-60 cells were sequentially labeled with the thymidine analogues iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU). The labeling index (LI), the duration of S-phase (Ts) and the total cell cycle time (Tc) were measured immediately. It was therefore possible to predict the next time when the single versus double labeled cells would re-enter the S-phase. In our study, the Tc was calculated to be 20 hours. The third label, tritiated thymidine (3HTdR), was introduced at the predicted time of 20 hours to confirm the validity of the previously calculated Tc. The actual percentage of cells which were labeled by (3HTdR) was very similar to the predicted value. We conclude, therefore, that the calculated cell cycle time correlated well with the actual cell cycle time, at least in a controlled in vitro culture system. This novel triple label method validates our double-label technique developed for cell cycle measurements.

High expression of transforming growth factor-beta long cell cycle times and a unique clustering of S-phase cells in patients with acute promyelocytic leukemia.

Expression of transforming growth factor-beta (TGF-beta), which inhibits the proliferation of hematopoietic progenitors, was investigated simultaneously with cell cycle characteristics in 63 bone marrow biopsies from 23 cases with acute promyelocytic leukemia (APL). Bromodeoxyuridine (BrdU) was administered to every patient (17 newly diagnosed) for determination of the labeling index (LI) and the durations of S-phase (Ts) and the cell cycle (Tc) of leukemic promyelocytes. APL cases had lower LI both in the bone marrow aspirate (6.1% v 11.4%, P = .008) and biopsy (21.1% v 28.0%, P = .001) and longer Tc (93.6 hours v 56.0 hours, P = .002) when compared with other French-American-British subtypes. TGF-beta expression (detected by a monoclonal anti-TGF-beta 2/beta 3 antibody) was dramatically high, especially in interstitial areas of the biopsies. S-phase cells were found as geographically restricted islands of proliferation (GRIPs) in 20 of 22 cases. Weekly biopsies showed an increment in TGF-beta on day 7 of therapy in 13 of 17 cases, while in vivo differentiation was noted in 9 of 15. We conclude that the presence of high TGF-beta expression may explain the biologic basis for the slowly cycling nature of leukemic promyelocytes in APL as well as the unique clustering of S-phase cells observed in GRIPs.

In situ cell cycle kinetics in bone marrow biopsies following sequential infusions of IUdR/BrdU in patients with hematopoietic malignancies.

Examination of the proliferative characteristics of myeloblasts was undertaken in situ in bone marrow (BM) biopsies of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) following sequential infusions of iodo- (IUdR) and bromodeoxyuridine (BrdU). The ability to identify S-phase cells which have incorporated both or either one of the labels in vivo by using two monoclonal antibodies in vitro permitted the measurement of labeling index (LI) and durations of S-phase (Ts) and the total cell cycle (Tc) both from the BM aspirates and biopsies. While the LI is 2-3 times higher in biopsies, Ts and Tc are fairly comparable in the two samples in 8/10 cases (p = 0.02 and 0.003 respectively). Advantages associated with the determination of cell cycle parameters in BM biopsies have been discussed at length.