REJUVENATION RESEARCHVolume 8, Number 1, 2005© Mary Ann Liebert, Inc.

Gerontomodulatory and Youth-Preserving Effects ofZeatin on Human Skin Fibroblasts Undergoing

Aging In Vitro

SURESH I.S. RATTAN1 and LAKSHMAN SODAGAM1,2

ABSTRACT

Our studies have shown that zeatin, (6-[4-hydroxy-3-methyl-but-2-enylamino]adenine), a cy-tokinin plant growth factor, has gerontomodulatory, youth preserving and anti-aging effectson serially passaged human adult skin fibroblasts undergoing aging in vitro. There were noimmediate negative or toxic effects in terms of cell attachment, cell proliferation, cell survival,cytoskeletal organization, and cellular growth by treatment with zeatin concentrations be-tween 1 and 200 �M. During long-term treatment, cells could be maintained throughout theirreplicative lifespan in the presence of 40, 80, and 200 �M zeatin, but the optimal concentra-tion of zeatin’s anti-aging and youth preserving effects was found to be 80 �M. Life-long se-rial passaging of human skin fibroblasts in the presence of zeatin resulted in the preventionof cell enlargement, reduction of intracellular debris, prevention of actin polymerization, andenhancement of cellular ability to decompose hydrogen peroxide and to cope with ethanoland oxidative stresses. Most importantly, anti-aging and beneficial effects of zeatin were ob-served without any induction of additional cell proliferation or an increase in the maximumproliferative capacity, thus ruling out any potentially harmful and carcinogenic effects.

46

INTRODUCTION

THE HAYFLICK SYSTEM of serially passagednormal diploid cells undergoing cellular

aging in vitro is a well established experimen-tal model system used extensively in biogeron-tological and anti-aging rejuvenation research.1Some recent examples of screening of suchcompounds and their successful developmentinto various cosmeceutical and neutriceuticalproducts include �-alanyl-L-histidine dipep-tide (carnosine),2,3 and N6-furfuryladenine orkinetin.4 Of these, the anti-aging and beneficialeffects of kinetin on human skin fibroblasts4

and on fruitflies were originally reported byus.5,6 These studies were followed by our de-tailed analysis of the molecular pathwaysthrough which kinetin appears to bring aboutits biological effects. For example, we hadshown that kinetin prevents and reduces theFenton-reaction mediated oxidative damage toDNA7 and inhibits the glycoxidation-mediateddamage to proteins.8 There is some evidencethat kinetin makes a complex with Cu,Zn-su-peroxide dismutase (SOD) and can indirectlyenhance catalase and glutathione peroxidaseactivities in order to counteract hydrogen per-oxide (H2O2) stress.9,10

1Laboratory of Cellular Ageing, Danish Centre for Molecular Gerontology, Department of Molecular Biology, Uni-versity of Aarhus, Aarhus, Denmark.

2Current address: Senetek PLC, Science Park, Aarhus, Denmark.

Since kinetin is a member of the cytokiningroup of plant growth factors,10,11 it is impor-tant to find out whether the anti-aging and an-tioxidative effects of kinetin can be observedfor other cytokinins also. Out of several nat-ural and synthetic cytokinins, 6-[4-hydroxy-3-methyl-but-2-enylamino] adenine, commonlyknown as zeatin, is one of the most widelystudied cytokinins with respect to its growthmodulatory and anti-senescence effects inplants.11 Although kinetin was the first cy-tokinin to be isolated in 1955 from the auto-claved samples of Sperm Whale DNA,12 it wasconsidered to be a thermally rearranged prod-uct, until our demonstration of the natural oc-currence of kinetin in DNA and cell extracts.13

Zeatin, on the other hand, was purified fromimmature kernels of the corn, Zea mays,14 andwas later identified to be present in the tRNAof a wide variety of organisms.11,15 Figure 1shows the chemical structure of zeatin, whichoccurs in two forms, trans and cis, but the cy-tokinin activity of zeatin is mainly attributed toits more stable trans form.11,16 There is a largebody of information available regarding thebiosynthesis, activity, and degradation ofzeatin and zeatin riboside in bacteria, fungi andhigher plants.17–21 However, almost nothing isknown about the effects of zeatin on animaland human systems.

Here we report the results of our studies onthe short-term and long-term treatment of hu-man skin fibroblasts with zeatin, which showthat, as in the case of kinetin, zeatin also hasseveral beneficial and youth-preserving effectson human cells undergoing aging in vitro.

MATERIALS AND METHODS

Cell culture and serial passaging

Primary cultures of normal diploid adultskin fibroblasts, designated ASF-2, were estab-lished from mammary skin biopsies obtainedfrom a healthy young female donor as de-scribed before.22–24 Cells were grown in T25plastic flasks (growth area 25 cm2, Cellstar,Germany, and Sarstedt Inc, USA) in an incu-bator at 37°C, 5% CO2, atmospheric oxygenconditions, and 95% humidity, in Dulbecco’sminimum essential medium (DMEM; BioWhittaker, Verviers, Belgium) supplementedwith 10% fetal calf serum (Biological Industries,Beit Haemek, Israel), 400 �M glutamine and100 U/mL penicillin/streptomycin (BioWhit-taker). This final medium is referred to as the“complete medium.” In the near-confluentstate, cells were trypsinized and subcultured ata 1:4 or 1:2 split ratio repeatedly until the endof their proliferative capacity in vitro. Fullrecords of the input and output of number ofcells was kept by counting the cell numbers us-ing a Coulter counter. Cumulative populationdoubling level (CPDL) achieved after serialpassaging was determined in accordance withthe well-established protocols for the Hayflicksystem of replicative senescence.22–24 Frequentchecks for mycoplasma (tested by fluorescentDNA staining by Hoechst 33258) showed thatthe cultures were free from mycoplasma infec-tion throughout their replicative lifespan.

Zeatin treatment

Trans-zeatin (6-[hydroxy-3-methyl-but-2-eny-lamino] adenine), molecular weight 219.25,99.5% purity, was purchased from OleChemimLtd (Czech Republic). Since zeatin is onlyslightly soluble in water, a stock solution (8mM) was prepared by dissolving zeatin in aminimum amount of 1N HCl (at about 30 mgzeatin per mL 1N HCl), followed by its appro-priate dilution in Hanks’ buffer, and filter ster-lization through 0.2-�m sterile filters. The stocksolution was stored in fridge at 4°C, and wasused for experiments by diluting it in the com-plete medium. Similarly, 8 mM stock solutionof zeatin riboside ([9-(�-D-Ribofuranosyl]-trans-zeatin; molecular weight 351.4, purchased from

ANTI-AGING EFFECTS OF ZEATIN 47

FIG. 1. Chemical structures of trans and cis zeatin.

Sigma Aldrich) was prepared. Pilot tests wereperformed to rule out any effects of the solventHCl by testing its maximum equivalent con-centrations (0.4% in 8 mM stock solution) onthe attachment, survival and growth of ASF-2cells. No biological effects of HCl could be de-tected, and therefore, no further controls withHCl were included in experiments on zeatinand zeatin riboside within the test concentra-tions (between 1 and 500 �M).

Cell attachment, survival, and short-term growth

In order to rule out any immediate negativeeffects of zeatin treatment, the number of cellsattached to the growth surface of cell cultureflasks was determined. An equal number of cells(about 10,000 cells per cm2 of growth area) wereseeded in multiple wells in 24-well tissue cultureplates (growth area 1.9 cm2) with or without dif-ferent concentrations of the test compound. Af-ter 6 h, the number of attached cells were countedin treated and untreated cells after trypsinisationand cell counting as described above.

Cell survival was measured by the 3-(4,5 di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide (MTT) assay. About 16,000 cells were seededper well in a 96-well plate 24 h before the exper-iment. Cells were then treated with different con-centrations of zeatin or zeatin riboside for 3 days,after which MTT (Sigma, M2128) was added at0.5 mg/mL in medium. After 4 h, MTT was re-moved and isopropanol and HCl were added todissolve the MTT crystals for 12–16 h. The ab-sorbance at 595 nm was measured using a spec-trophotometer. Data for “3-day cell survival” wasstatistically analyzed by Student’s t-test.

Short-term growth experiments were per-formed using freshly prepared cell suspensionsfrom mass cultures of early passage ASF-2 cells.About 10,000 cells were seeded into six sets of24-well plates and treated with various con-centrations of zeatin. The number of cells werecounted after different days of treatment in twowells by following the normal method of celltrypsinization and counting using a Coultercounter. The third well in each test categorywas fixed in cold methanol and stained withGiemsa stain for permanent record and forphotography. The experiment was carried onuntil the cultures became fully confluent andno further growth of cells was possible.

Long-term serial passaging of ASF-2 cells wasperformed in at least three parallel cultures in T25flasks with or without the addition of 40, 80, and200 �M trans-zeatin until the end of the replica-tive lifespan. Cultures were considered to be ir-reversibly senescent after complete cessation ofcell growth during a period of more than 1month after last subculturing and showing morethan 95% cells with senescence-specific �-galac-tosidase staining.26 During serial passaging,fresh complete medium with or without zeatinwas replaced on twice a week basis throughoutthe replicative lifespan of ASF-2 cells.

Other methods

Apoptosis assay. The extent of apoptosis intreated and untreated cell cultures was deter-mined by DAPI-staining of DNA, using fluo-rescence microscope. Staurosporine (0.2 �M)was used as an inducer of apotosis for stan-dardising the method.

DNA synthesis assay. Proportion of cells undergo-ing DNA duplication, and thus entering the nextround of cell division, was determined by labellingthe cells with bromodeoxyuridine, using a com-mercially available kit from Roche, Germany.

Actin staining. The pattern of cytoskeletal actinstaining was studied by staining the cells withfluorescent ligand FITC-labelled phalloidin, us-ing a fluorescence microscope.25

�-gal staining. Senescence-specific �-galactosi-dase staining at pH 6.0 was performed follow-ing Dimri’s method.26

Hydrogen peroxide (H2O2) decomposition assay.H2O2 decomposition assay in vitro, which is ameasure of the general antioxidant ability ofcells combining catalase and glutathione per-oxidase activities, was determined by spec-trophotometeric method.27

RESULTS

Immediate and short-term effects of zeatin

In order to rule out any immediate harmfuleffects of zeatin on human skin fibroblasts, abattery of tests were performed on young cells

RATTAN AND SODAGAM48

(early passage, less than 30% lifespan com-pleted) and on senescent cells (late passage,more than 80% lifespan completed). Table 1summarizes these results which showed thatzeatin concentrations up to 200 �M had no neg-ative effects on the cell attachment frequency,on 3-day cell survival, on BrdU-labeling index,on the number of senescence-specific �-galac-tosidase positive cells and on the number ofspontaneously apoptotic cells. However,higher concentrations of zeatin (250 �M, 500�M, 1 mM, and 2 mM) affected the 3-day cellsurvival significantly with less than 15% cellssurviving at the highest concentrations.

These observations were further confirmedby one step growth analysis during a period ofup to 11 days. Figure 2 shows that ASF-2 cellscould grow and proliferate at almost similarrates in the absence or presence of zeatin at con-centrations up to 80 �M. However, there wasa slight slowing down of growth in 200 �Mzeatin-treated cultures, but a significant slow-ing down of growth in 500 �M zeatin-treated.Therefore, for all long-term experiments on the

effects of zeatin on ASF-2 cells, a maximumconcentration of up to 200 �M was used.

Long-term effects of zeatin treatment

Replicative lifespan, morphology, and cell yield.Figure 3 shows the longevity curves for ASF-2cells treated with various doses of zeatinthroughout their replicative lifespan in vitro. Inthis series of experiments untreated controlASF-2 cells achieved CPDL 27.85 before be-coming irreversibly growth arrested after 286days of continuous serial passaging. This muchreplicative lifespan of 27.85 CPDL in this seriesof experiments is here considered as 100% life-span completed. Under the same conditions,ASF-2 cells continuously grown in the presenceof 40, 80, and 200 �M zeatin underwent 28.04,28.27, and 27.96 CPDL, respectively.

Age-related changes in cellular morphology,cell size, cytoskeletal organization, and variousbiochemical parameters are well establishedmarkers of youthfulness and senescence in theHayflick system of cellular aging in vitro. In the

ANTI-AGING EFFECTS OF ZEATIN 49

TABLE 1. EFFECTS OF SHORT-TERM TREATMENT OF EARLY PASSAGE

YOUNG HUMAN SKIN FIBROBLASTS WITH ZEATIN

Characteristic Untreated cells Zeatin-treated cells

Attachment frequencies 80–90%0 80–90%0S-phase–positive cells 75–80%0 75–80%03-day cell survival 90–100% 90–105%�-gal–positive cells �5% �5%Apoptotic cells �5% �5%

FIG. 2. Effect of zeatin on one-step growth of human skin fibroblasts.

present series of experiments, some of thesechanges were compared between untreatedand zeatin-treated ASF-2 cells throughout theirreplicative lifespan. Figure 4 shows a morpho-logical comparison between early passageyoung cultures (�30% lifespan completed) andlate passage senescent cultures (�90% lifespancompleted) grown in the absence or presenceof 40, 80, and 200 �M zeatin. In untreated con-trol cultures, there was a dramatic change inthe morphology of cells from thin, long, spin-dle shaped cells arranged in fingerprint-like ar-rays (Fig. 4A) to highly enlarged, disorganized,vacuolated and sometimes, multinucleatedsenescent cells full of intracellular debris (Fig.4B). In comparison, whereas zeatin treatedyoung cells appeared to be very much similarto the untreated cells (Fig. 4C,E,G), late passagesenescent cells treated with zeatin had muchbetter appearance and structural integrity thanuntreated cells (Fig. 4D,F,H). There was a sig-nificant preservation of youthful morphologyin cells treated with 40 and 80 �M zeatin, butthe most dramatic effects could be observed incells grown in the presence of 80 �M zeatin(Fig. 4F). Zeatin-treated cells maintained theirspindle shape and arrangement in fingerprint-like arrays and avoided any significant en-largement, vacuolation and accumulation of in-tracellular debris.

Since one of the main youth preserving ef-fects of zeatin was the maintenance of youngmorphology of cells, this was further con-firmed by the data obtained for cell yield in

confluent cultures, which is an indirect mea-sure of cell size (Fig. 5). Early passage untreatedyoung confluent cultures had a cell yield in therange of 15–20,000 cells per cm2, which wasprogressively reduced to about 6–9,000 cellsper cm2 in the mid-passages, and to about 5,000cells per cm2 in senescent cultures. In compar-ison, cell cultures maintained in the presenceof 40 and 80 �M zeatin generally had a highercell yield both at early passages (more than15,000 cells per cm2) and at late passages (morethan 10,000 cells per cm2).

Cytoskeletal organization. Alterations in the cy-toskeletal organization are one of the crucialage-related changes during cellular aging invitro. Figure 6A shows three typical fluores-cence staining patterns seen for actin in ASF-2cells. These types are termed here as “actinstaining type (AST) 1, 2, and 3,” respectively.AST1 is the diffused staining pattern of actinhomogenously dispersed throughout the cellwith little or no polymerization. AST2 is themixed and heterogeneous pattern of actinstaining where both a diffused distribution andstring-like polymerized pattern of actin can beobserved. AST3 is the highly polymerized rod-like staining pattern observed most commonlyin enlarged and senescent cells. These threepatterns of actin staining can be observed in cellcultures at all passage levels, but their relativeproportions change during serial passagingand cellular aging in vitro. Visual identificationand counting of the number of cells with dif-

RATTAN AND SODAGAM50

FIG. 3. Effect of zeatin on long-term growth and longevity of human skin fibroblasts.

ferent ASTs, using a flourescence microscope,shows that untreated control cultures at earlypassage had 68% AST1 cells, and 32% AST2cells with no detectable AST3 cells (Fig. 6B). Onserial passaging, the proportion of differentAST in late passage senescent cultures changedto 8% AST1, 67% AST2 and 32% AST3, respec-tively (Fig. 6C).

Zeatin treatment of ASF2 cells significantlyprevented these age-related alterations in actin

polymerization. Figure 6B shows that earlypassage cultures grown in the presence of 40,80, and 200 mM zeatin had significantly in-creased proportion of AST1 cells (78–94%) andreduced proportion of AST2 cells (5–20%). Sim-ilar trends in the maintenance of youthful actinstaining patterns could be seen in late passagesenescent ASF-2 cultures maintained in differ-ent concentrations of zeatin throughout theirreplicative lifespan in vitro. Figure 6C shows

ANTI-AGING EFFECTS OF ZEATIN 51

FIG. 4. Effect of zeatin on the morphology of serially passaged cultures of human skin fibroblasts. Original magni-fication, �20.

that zeatin-treated late passage cultures hadhigher proportion of youthful AST1 cells(30–60% vs. 8% in controls) and reduced pro-portion of near senescent AST cells (35–55% vs.67% in controls), and senescent AST3 cells(2–10% vs. 32% in controls).

H2O2 decomposing ability. Antioxidative effectsof zeatin were tested by determining the H2O2decomposing ability of crude extracts preparedfrom treated and untreated young and senes-cent ASF2 cells. Figure 7 shows that early pas-sage young cells grown for 3 PD in the pres-ence of different concentrations of zeatin hadsignificantly increased ability to decomposeH2O2. However, this increase was not strictlydose-dependent. For example, whereas earlypassage young ASF-2 cells treated with 40 and80 �M zeatin had an increase in their H2O2 de-composition ability by 59% and 236%, respec-tively, this increase was only about 27% in 200�M–treated cells. Serial passaging of ASF-2cells did not result in a decrease in H2O2 de-composing ability of late passage senescentcells as compared with early passage youngcells, and zeatin-treated senescent cells main-tained higher H2O2 decomposing abilities evenat the end of their lifespan in vitro.

In addition to the above experiments, we hadalso determined the effects of zeatin on the in-duction of apoptosis both on its own and in thepresence of other stresses, such as ethanol andH2O2. Table 2 gives the results of the effects of

zeatin treatment on the inhibition of apoptosisin young and senescent cells. There was no ad-ditional induction of apoptosis by zeatin treat-ment (40–200 mM) in young and senescent cells(1–5% spontaneous apoptosis). On the otherhand, whereas 10% ethanol or 500 mM H2O2treatment for 3 days induced apoptosis in morethan 80% young and senescent cells, zeatin-treated cells resisted these stresses and only10–15% cells became apoptotic (Table 2).

DISCUSSION

The results obtained in this series of exper-iments provide evidence for zeatin as anotherexample of cytokinins having gerontomodu-latory, youth preserving and anti-aging ef-fects on human skin fibroblasts. Previously,work done in our labs had shown that kinetinhad powerful anti-aging effects on humanskin fibroblasts.4 Those studies were followedup by a thorough and systematic testing ofkinetin for its suitability and marketing as acomponent of numerous skin care prod-ucts.28,29 (also see www.senetekplc.com). Ournew data on zeatin suggest that it may be an-other cytokinin with a potential for develop-ment into anti-aging and health care prod-ucts. Similar studies on other cytokinins andtheir derivatives may discover other com-pounds with applications in aging preven-tion, intervention and therapy.

RATTAN AND SODAGAM52

FIG. 5. Effect of zeatin on cell yield of serially passaged cultures of human skin fibroblasts.

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Our studies on the short-term and immedi-ate effects of zeatin have ruled out any negative and toxic effects in terms of cell attachment, cell proliferation, cell survival, cytoskeletal organization, and cellular growthup to 200 �M concentration. Higher concen-trations of zeatin do affect cell survival andgrowth, but it should be pointed out that suchnegative effects at high doses can be observeduniversally for antioxidants, hormones, growth

factors, and other chemical entities. The im-portant point is to determine the biologicallyeffective and beneficial window of concentra-tion, which in the case of zeatin appears to bebetween 40 and 200 �M, and is comparable tothe previously reported range for kinetin.

The optimal concentration of zeatin for itsanti-aging and youth preserving effects on hu-man skin fibroblasts is between 40 and 80 �M,which prevents, reduces and delays the onsetof age-related cellular and biochemical alter-ations. The main biomarkers of cellular agingthat we have studied in this series of experi-ments include cell enlargement, accumulationof cellular debris, increased actin polymeriza-tion, decreased antioxidative ability and re-duced stress resistance. Zeatin treatment of hu-man cells not only slows down their aging interms of modulating above mentioned bio-markers, these effects are achieved without in-terfering with the genetic regulation of theircellular proliferative capacity or the Hayflicklimit. This fact is underlined by our observa-tions that zeatin treatment of human skin fi-broblasts neither induces cell proliferation on ashort-term basis nor does it increase the maxi-mum proliferative lifespan (CPDL) on a lifelong basis. Therefore, one could infer thatzeatin does not have any potentially harmfuland carcinogenic effects on human cells and isnot expected to upset any genetic mechanismsof proliferation regulation, such as telomereloss and DNA methylation.30,31

RATTAN AND SODAGAM54

TABLE 2. EFFECT OF ZEATIN ON THE INDUCTION OF

APOPTOSIS IN THE PRESENCE OF OTHER STRESSES

Percentage of apoptotic cellsa

Treatment Young cells Senescent cells

No treatment 1–5 1–5Ethanol (10%) 85–90 25–30H2O2 (500 �M) 50–55 70–80

Zeatin 40 �M 1–4 1–4� Ethanol (10%) 75–80 65–75� H2O2 (500 �M) 5–10 10–15

Zeatin 80 �M 1–5 1–4� Ethanol (10%) 65–70 20–25� H2O2 (500 �M) 15–20 10–12

Zeatin 200 �M 1–5 1–4� Ethanol (10%) 70–80 20–25� H2O2 (500 �M) 15–20 10–15

aPercentage range of apoptotic cells was determined byfluorescence microscopic count of at least 500 cells in eachcondition in two separate experiments.

FIG. 7. Effect of zeatin on hydrogen peroxide decomposing ability of serially passaged human fiboroblasts.

The most dramatic effects of zeatin on hu-man skin fibroblasts observed in these studiesare in terms of maintaining the youthful ap-pearance of cells and enhancing their antiox-idative ability. Since age-related cell enlarge-ment and disorganization is mainly due to theaccumulation of debris consisting of oxida-tively and glyoxidatively modified pro-teins,32,33 one can assume that zeatin treatmentof cells affects this change either by enhancingthe turnover of abnormal proteins through pro-teasomal and lysosomal pathways and/or bypreventing the formation of abnormal proteinsduring aging. Direct evidence in support of anyof these mechanisms with respect to the modeof action of zeatin awaits further investigations.However, our previous studies on the molecu-lar mechanisms of action of kinetin have shownthat kinetin, which also has similar anti-agingaffects on human cells, protects DNA and pro-teins against oxidative and glyoxidative dam-age.7,8 Therefore, it will be interesting to findout whether zeatin also works through thesame antioxidative pathways of defence andmaintenance. There is some indication fromour observations on the enhanced ability ofzeatin-treated cells to decompose H2O2 thatzeatin stimulates cellular antioxidative path-ways involving catalase and glutathione per-oxidase. Further studies are required to fullyunderstand the antioxidative mode of action ofzeatin on human cells.

Another significant effect of zeatin on humanskin fibroblasts undergoing aging in vitro is theprevention of actin polymerization into rigidrod-like structures which influence intracellu-lar molecular flow and interactions.34,35 Earlypassage young cultures are comprised of a ma-jority of cells with homogenously distributeddiffused pattern of monomeric forms of actin,as visualized by staining pattern termed AST1described in the results above. Although onecan observe even in early passage culturessome AST3 type cells, it is the progressive shiftin their proportion from AST1 in young cul-tures to AST2 and 3 in late passage cultures thattypifies the cellular aging process. Several ofthe age-related alterations in cellular biochem-istry and physiology, such as protein synthe-sis, modification, localization and degradation,and entry into the cell cycle are negatively as-

sociated with the rigidity of the cytoskele-ton.34,35 The presence of zeatin in the cell culture medium significantly reduces this age-related shift in actin rigidity, which may havebeneficial effects on cellular functioning andperformance. However, it will be important toknow what happens to other components of thecytoskeleton, especially microtubules whichare involved in the regulation of cell cycle andcell proliferation. Furthermore, what effectszeatin has on the synthesis, modification andturnover of total and specific proteins, such ascollagen and other extracellular matrix compo-nents also remains to be determined. Our pres-ent series of experiments opens up a wholerange of important lines of investigation thatwill not only lead to attaining a complete un-derstanding of the biological effects and mo-lecular mechanism of action of zeatin, but alsowill facilitate identifying appropriate intracel-lular and extracellular targets where zeatin andother related compounds may have beneficialhealth care applications.

Since the effects of zeatin reported here arevery much similar to the previously reportedeffects of kinetin, some comments on a com-parison between kinetin and zeatin are war-ranted. Although studies performed on kinetinand zeatin are more than ten years apart andhence making any comparisons between theirefficacies cannot be definitive, it appears thatzeatin and kinetin are very much alike in theiranti-aging effects on human cells. However,there are a few important differences betweenkinetin and zeatin treatments. For example, ona short-term basis, human skin fibroblasts cantolerate higher concentration of zeatin (200 �M)as compared with 100 �M of kinetin. On a long-term basis, although cells can be maintained atup to 200 �M zeatin throughout their lifespan,the optimal anti-aging effects are seen at 80 �M,which is the same as in the case of kinetin. In-terestingly, most dramatic differences betweenzeatin and kinetin were observed when theirrespective ribosides are used. Whereas kinetin-riboside is highly toxic for cells even at 10 �Mconcentration,4 zeatin-riboside had no toxic ef-fects even up to 500 �M, as tested by 3-dayMTT survival assay (data not shown). At pres-ent, we have no data for the long term effectsof zeatin-riboside on human cells.

ANTI-AGING EFFECTS OF ZEATIN 55

Finally, from a mechanistic point, it is in-triguing that a DNA-based modified adenine(kinetin) and a tRNA-based modified adenine(zeatin) have similar anti-aging and youth pre-serving biological effects when supplied fromoutside to human skin fibroblasts. Further re-search on the molecular basis of the modes ofaction of kinetin, zeatin and other cytokininsand their derivatives is required to unraveltheir modes of action and to compare and con-trast their biological properties.

ACKNOWLEDGMENTS

Laboratory of Cellular Ageing is supportedby research grants from the Danish MedicalCouncil (SSVF), Danish Natural Science Coun-cil (SNF), and Senetek PLC.

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Address reprint requests to:Dr. Suresh I.S. Rattan

Department of Molecular BiologyUniversity of Aarhus

Gustav Wieds Vej 10-CDK-8000 Aarhus-C, Denmark

E-mail: rattan@mb.au.dk

Received: September 10, 2004Accepted: December 13, 2004

ANTI-AGING EFFECTS OF ZEATIN 57