Flow cytometric analysis of the cell cycle in polyamine‐depleted cells

Pohjanpelto, Pirkko; Nordling, Stig; Knuutila, Sakari

doi:10.1002/cyto.990160407

Cited by 23 publications

(18 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These data fit with observations that polyamines are necessary for the G1\S transition [34a,35] or the progression through S phase [36,37]. Decreased polyamine concentrations would then be expected to decrease mitoses, as seen here and in other systems [36,38]. Down-regulation of cellular polyamines appears to be necessary not only for cell growth but also for survival.…”

Section: Discussionsupporting

confidence: 86%

Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis

Tome

Fišer

Payne

et al. 1997

Biochemical Journal

View full text Add to dashboard Cite

DH23A cells, an α-difluoromethylornithine-resistant variant of the parental hepatoma tissue culture cells, express high levels of stable ornithine decarboxylase. Aberrantly high expression of ornithine decarboxylase results in a large accumulation of endogenous putrescine and increased apoptosis in DH23A cells when α-difluoromethylornithine is removed from the culture. Treatment of DH23A cells with exogenous putrescine in the presence of α-difluoromethylornithine mimics the effect of drug removal, suggesting that putrescine is a causative agent or trigger of apoptosis. Accumulation of excess intracellular putrescine inhibits the formation of hypusine in vivo, a reaction that proceeds by the transfer of the butylamine moiety of spermidine to a lysine residue in eukaryotic initiation factor 5A (eIF-5A). Treatment of DH23A cells with diaminoheptane, a competitive inhibitor of the post-translational modification of eIF-5A, causes both the suppression of eIF-5A modification in vivo and induction of apoptosis. These data support the hypothesis that rapid degradation of ornithine decarboxylase is a protective mechanism to avoid cell toxicity from putrescine accumulation. Further, these data suggest that suppression of modified eIF-5A formation is one mechanism by which cells may be induced to undergo apoptosis.

show abstract

Section: Discussionsupporting

confidence: 86%

Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis

Tome

Fišer

Payne

et al. 1997

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…In contrast, CHENSpm treatment produces a significant G 2 ͞M block concurrent with its cytotoxic activity. This effect by CHENSpm is somewhat unusual in that interference with polyamines metabolism and polyamine depletion generally produce a G 1 ͞S block if they have any measurable effect on the cell cycle at all (33,34).…”

Section: Discussionmentioning

confidence: 99%

The role of polyamine catabolism in polyamine analogue-induced programmed cell death

Woster

Yager

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

256

190

View full text Add to dashboard Cite

N 1 -ethyl-N 11 -[(cyclopropyl)methyl]-4,8,-diazaundecane (CPENSpm) is a polyamine analogue that represents a new class of antitumor agents that demonstrate phenotype-specific cytotoxic activity. However, the precise mechanism of its selective cytotoxic activity is not known. CPENSpm treatment results in the superinduction of the polyamine catabolic enzyme spermidine͞spermine N 1 -acetyltransferase (SSAT) in sensitive cell types and has been demonstrated to induce programmed cell death (PCD). The catalysis of polyamines by the SSAT͞polyamine oxidase (PAO) pathway produces H 2 O 2 as one product, suggesting that PCD produced by CPENSpm may be, in part, due to oxidative stress as a result of H 2 O 2 production. In the sensitive human nonsmall cell line H157, the coaddition of catalase significantly reduces high molecular weight (HMW) DNA (>50 kb) and nuclear fragmentation. Important to note, specific inhibition of PAO by N,N-bis(2,3-butadienyl)-1,4-butane-diamine results in a significant reduction of the formation of HMW DNA and nuclear fragmentation. In contrast, the coaddition of catalase or PAO inhibitor has no effect on reducing HMW DNA fragmentation induced by N 1 -ethyl-N 11 -[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not induce SSAT and does not deplete intracellular polyamines. These results strongly suggest that H 2 O 2 production by PAO has a role in CPENSpm cytotoxicity in sensitive cells via PCD and demonstrate a potential basis for differential sensitivity to this promising new class of antineoplastic agents. Furthermore, the data suggest a general mechanism by which, under certain stimuli, cells can commit suicide through catabolism of the ubiquitous intracellular polyamines.Programmed cell death (PCD) is a fundamental biological regulatory mechanism involving selective cell deletion. It is an active and irreversible process in which cells activate the intrinsic death program for their own demise. PCD is absolutely required for the natural development and homeostasis of tissues in complex multicellular organisms (1-4). Morphological characteristics of PCD include cell shrinkage, nuclear condensation and fragmentation, plasma and nuclear membrane budding, and apoptotic bodies (3). PCD is biochemically characterized by activation of nucleases that cleave chromosomal DNA into high molecular weight (HMW) and͞or low molecular weight oligonucleosomal DNA fragments (5). PCD can be induced by normal physiological processes and by multiple nonphysiological stimuli, including oxidative stress and chemotherapeutic agents (4-7).The polyamines spermidine and spermine and their diamine precursor putrescine are intracellular cationic molecules that are essential for cell proliferation and differentiation (8, 9). The intracellular concentration of these ubiquitous molecules is highly regulated by the polyamine metabolic pathway, which influences the synthesis, degradation, uptake, and excretion of the cations (9). High ornithine decarboxylase (ODC) activity, the first rate-limiting step of polyami...

show abstract

“…This early effect has been possible to detect due to the sensitivity of the flow cytometry method used with BrdUrd. Earlier reports have dealt with impaired DNA replication after several days of inhibitor treatment (Anehus et al, 1984;Westin et al, 1991 ;Pohjanpelto et al, 1994). Pohjanpelto and Hiiltta (1996) have shown that polyamine depletion reversibly blocks synthesis of high-molecutar-mass DNA and leads to accumulation of short DNA pieces, suggesting a role for polyamines in joining the Okazaki fragments.…”

Section: Discussionmentioning

confidence: 99%

Ordered Cell Cycle Phase Perturbations in Chinese Hamster Ovary Cells Treated with an S‐Adenosylmethionine Decarboxylase Inhibitor

Fredlund

Oredsson

1997

European Journal of Biochemistry

View full text Add to dashboard Cite

The polyamines putrescine, spermidine, and spermine are needed for normal cell cycle progression and polyamine-depleted cells cease to proliferate. We have investigated cell cycle perturbations in Chinese hamster ovary cells seeded in the presence of 4-amidinoindan-I-one 2'-amidinohydrazone (CGP 48664), a potent inhibitor of S-adenosylmethionine decarboxylase, an enzyme which is essential for the synthesis of spermidine and spermine. At 9 h and at 1, 2, and 3 days after seeding, cells were labelled with the thymidine analog bromodeoxyuridine (BrdUrd) for 30 min, after which the BrdUrd-containing medium was removed and the cells were allowed to progress through the cell cycle in BrdUrd-free medium before sampling (post-labelling time). Using flow cytometry, coupled with an indirect immunofluorenscence technique, utilizing monoclonal anti-BrdUrd and secondary fluorescein-isothiocyanate-conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. By investigating the movement of BrdUrd-nonlabelled G , cells into S phase during the post-labelling time, a measure of the G,/S transition was obtained. The time of appearence in G , of BrdUrd-labelled cells which had divided was used to monitor the length of the G,+M phase. A measure of the S phase length (DNA synthesis time) was obtained by monitoring the progression of BrdUrd-labelled cells through S phase. CGP 48664-induced spermine depletion significantly increased the length of the S phase already 9 h after seeding cells in the presence of the inhibitor. No effects 011 the G,/S transition or on the length of the G,+M phase were observed until 2 days after seeding.

show abstract

Flow cytometric analysis of the cell cycle in polyamine‐depleted cells

Cited by 23 publications

References 28 publications

Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis

Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis

The role of polyamine catabolism in polyamine analogue-induced programmed cell death

Ordered Cell Cycle Phase Perturbations in Chinese Hamster Ovary Cells Treated with an S‐Adenosylmethionine Decarboxylase Inhibitor

Contact Info

Product

Resources

About