ФЕНОМЕН "СТАЦИОНАРНОГО СТАРЕНИЯ" КЛЕТОЧНЫХ КУЛЬТУР: ТРИДЦАТЬ ЛЕТ ФЕНОМЕН "СТАЦИОНАРНОГО СТАРЕНИЯ" КЛЕТОЧНЫХ КУЛЬТУР: ТРИДЦАТЬ ЛЕТ СПУСТЯСПУСТЯ

А.Н.ХохловА.Н.Хохлов

Сектор эволюционной цитогеронтологии, биологический факультет МГУ имени М.В.Ломоносова, Москва, РоссияСектор эволюционной цитогеронтологии, биологический факультет МГУ имени М.В.Ломоносова, Москва, Россия

khokhlov@mail.bio.msu.rukhokhlov@mail.bio.msu.ru

Cell SenescenceCell Senescence

In culture experiments а la Hayflick, researchersestimate the replicative capacity of cells by counting the number of times a culturecan be split before the cells stop dividing. Trypsin is added to free the cells from the extracellular matrix, and cultures are grown until cells carpet the floor of the flasks. This protocol spawned the legendary Hayflick limit (from Hopkin, 2001).

• Hayflick’s phenomenon (IOW - “cell senescence”; unfortunately, last years this term, in my opinion, has rather often been used incorrectly by those who relate it to so called DNA damage response) – limited normal cell proliferation in vitro and in vivo (Population Doubling Level limit)• From the first to the last passage of “senescing” cells they change in the same way as cells of aging multicellular organism• We cannot explain why we age just with the help of the Hayflick’s phenomenon (say, the most important organs – brain and heart – contain a lot of postmitotic cells like neurons or cardiomyocytes) • The Hayflick’s phenomenon is defined by a set of various correlations only

However, now we know how the Hayflick’s phenomenon is realized (thanks to Alexei

Olovnikov!)

Хохлов А.Н., Чиркова Е.Ю., Ушаков В.Л.Хохлов А.Н., Чиркова Е.Ю., Ушаков В.Л.

Использование стационарных культур Использование стационарных культур для изучения механизмов клеточного для изучения механизмов клеточного

старениястарения

В сб.: Первый съезд Белорус. общества В сб.: Первый съезд Белорус. общества геронтологов и гериатров. Тез. докл. геронтологов и гериатров. Тез. докл.

Минск, 1983, 188-189Минск, 1983, 188-189

THEORY OF AGINGCell proliferation restriction is the main cause of accumulation in cells of DNA

damage inducing, in turn, other damageand, as a result, cell death

PROPOSED MODELStationary cell cultures (it is assumed

that under in vitro cell proliferationrestriction cells accumulate the damage

similar to those in cells aging in vivo)

PRIMARY CAUSE OF AGINGDNA damage

ALL OTHER AGE-DEPENDENTCHANGES, AGE DISEASES

INCREASE OF ORGANISM’SDEATH PROBABILITY

Influence on this stageallows to find out whetherthe factor investigated isgeroprotector or geropromoter

Influence on this stageby geroprotector or geropromoter modulatesthe rate of aging onlytemporarily

Cell aging is the accumulation in a cell population of various

types of damage identical to the

damage arising in senescing

multicellular organism

Schematic representation of cultured cell “stationary phase aging” phenomenon

Cel

l d

ensi

ty (

rela

tive

un

its)

Time after subcultivation (days)

Survival curve of Chinese hamster stationary cell culture (solid line – experimental points; dotted line – approximation by the Gompertz equation;

straight line – mortality rate changes with time)

??

Telomeres and TelomeraseTelomeres and Telomerase

«Age» changes shown to really occur in stationary

cell cultures• Accumulation of DNA breaks and DNA-protein cross-

links• Accumulation of 8-oxo-2’-deoxyguanosine in DNA• Inhibition of poly-ADP-ribosylation of chromatin proteins• DNA demethylation• Changes of spontaneous sister chromatid exchange

level• Plasma membrane changes• Nuclear structure modulations• Decrease of the rate of mitogen-stimulated cell cycling

and of cell colony-forming ability

«Stationary phase aging» experiments have been already

carried out on:

• Normal and transformed human and animal cells

• Plant cells

• Bacteria

• Mycoplasmas

• Yeasts

BacteriaBacteria

YeastsYeasts

20122012

Thanks for your attention and patienceThanks for your attention and patience, , sincerely yours,sincerely yours,

AN KhAN Khokhlovokhlov

If you want to make God laugh – If you want to make God laugh – describe to Him your theory of describe to Him your theory of

agingaging

Naturally, anybody from the Naturally, anybody from the audience has the right to object to audience has the right to object to me. And I can certainly agree with me. And I can certainly agree with

him.him.

But then we'll both be wrong - But then we'll both be wrong - ..