Usefulness of PKH fluorescent labelling to study leukemic cell proliferation with various cytostatic drugs or acetyl tetrapeptide--AcSDKP.
BACKGROUND
PKH67 labelling was compared for classical proliferation assessment (using S phase evaluation) to analyse the cell proliferation of 29 AML patients treated or not with various drugs. Among these drugs, the effect of tetrapeptide AcSDKP or AcSDKP-NH2 on AML cells, stimulated or not by cytokines, was also evaluated in order to determine (i) if AcSDKP was able to inhibit blast cell proliferation as it inhibits haematopoietic progenitors (ii) if AcSDKP-NH2 was more stable than AcSDKP with FBS.


METHODS
For PKH labeling, cells were suspended in Diluent C, and rapidly admixed with PKH67 solution at 20 microM PKH67. Staining was stopped by addition of FBS.


RESULTS
A good correlation between PKH67 labelling and bromodeoxyuridine incorporation was obtained first with 6/9 patients for control cells, then for 11/17 AML patients treated with classical antileukemic drugs (among whom 4 were also treated with AcSDKP). The effect of AcSDKP was also studied on 7 patients. The discrepancy between both methods was essentially due to an accumulation of cells into different cycle phases measured by BrdUrd incorporation secondary to drug action and PKH67 labelling which measured the dynamic proliferation. This last method allows identifying resistant cells which still proliferate. AcSDKP or AcSDKP-NH2 induced a decrease of leukemic cell proliferation in 5/7 patients when cytokines were added (in order to stimulate proliferation) one day after tetrapeptide AcSDKP or AcSDKP-NH2. No effect on proliferation was noted when cytokines were added to AcSDKP-NH2.


CONCLUSIONS
PKH67 labelling method is a powerful tool for cell proliferation assessment in patients with AML, even in cells treated by various drugs.
Boutonnat
Jean
J
Laboratoire de Dynamique Cellulaire, EPHE, IFRT 130, CNRS UMR 5525, 38706 La Tronche cedex, France. jean.boutonnat@ujf-grenoble.fr
Faussat
Anne-Marie
AM
Marie
Jean-Pierre
JP
Bignon
Jérôme
J
Wdzieczak-Bakala
Johanna
J
Barbier
Magali
M
Thierry
Josiane
J
Ronot
Xavier
X
Colle
Pierre-Emmanuel
PE
D000677:Amsacrine / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D049109:Cell Proliferation; D003561:Cytarabine / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D003630:Daunorubicin / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D006843:Hydrocarbons, Chlorinated / Q000494:pharmacology; D007938:Leukemia / Q000473:pathology*; D015470:Leukemia, Myeloid, Acute / Q000473:pathology; D008856:Microscopy, Fluorescence / Q000379:methods*; D009842:Oligopeptides / Q000494:pharmacology*; D009930:Organic Chemicals / Q000494:pharmacology; D011837:Radiation-Protective Agents / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D005456:Fluorescent Dyes; D006843:Hydrocarbons, Chlorinated; D009842:Oligopeptides; D009930:Organic Chemicals; C451241:PKH67; D011837:Radiation-Protective Agents; D000677:Amsacrine; D003561:Cytarabine; D005047:Etoposide; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; C004986:chlorbenside; D001973:Bromodeoxyuridine; C058504:goralatide; D003630:Daunorubicin