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Importance of circadian rhythms in animal cell cultures
Camp. Biochem. Phvsiol. Vol. 76A, No. 4, pp. 113-776, 1983
Printedin Great Biitain
c
030%9629/83 $3.00 + 0.00 1983 Pergamon Press Ltd
MINIREVIEW
IMPORTANCE OF CIRCADIAN RHYTHMS ANIMAL CELL CULTURES
IN
RANJANA KADLE and C. EDGAR FOLK, JR. Department
of Physiology
and Biophysics, The University of Iowa, Telephone (3 19) 353-4152
Iowa City. IA 52242, USA,
Abstract-l. Circadian rhythms are a characteristic feature of many cell and organ cultures. 2. Such rhythms may be important in the interpretation of data from cells in culture. 3. More examples of circadian rhythms in tissue culture are badly needed to understand phenomena. However, they will only be expressed under optimum and well-understood conditions.
INTRODUCTlON
The importance of circadian rhythms (CR) in cellular function is sometimes overlooked when working with culture systems. Much of the data from such systems is interpreted without considering the possibility of some effect being due to circadian variations. The presence of CR is a specific characteristic of whole organisms (unicellular as well as multicellular). Although the observations made on unicellular organisms cannot be extended easily to isolated cells derived from more complex multicellular animals, it is plausible that the basic unit of the circadian clock resides in these isolated individual cells. A number of studies have been conducted to determine whether CR are exhibited by cells or organs when isolated from the body. In this paper we review some of these results, and we stress the importance of exploring for the presence of such rhythms whenever culture systems are used. EVIDENCE FOR THE PRESENCE OF CR IN ORGAN AND CELL CULTURES
There is evidence for CR in cultures of: (1) the eye of A&&, (2) pineal glands and cells cultured from these glands. both avian and mammalian, (3) nervous tissue of insects, (4) adrenal glands and cells cultured from these, (5) isolated heart and heart cell networks and (6) liver cells. The isolated eye of the sea hare, Apl_vsia culifornicu, displays CR of large amplitude in the compound action potentials (CAP) of the optic nerve impulses (Jacklet, 1969: Eskin et ul., 1982). These can be recorded Sor up to 2 weeks in constant darkness (Jacklet. 1974). The peak of the CAP activity, in constant darkness, for animals entrained to a LD 12: I2 cycle is at the projected dawn, while the minimum is at the projected dusk (Rothman and Strumwasser, 1976). In constant darkness the correspondence of the activity cycles to the cycle to which the animals were entrained decreased gradually. They acquired a free-running rhythm. 773
this
The cultured chick pineal gland displays a CR in the secretion of the hormone melatonin. Maximum levels are observed during night-time and the minimum during daytime. The variation in the levels of melatonin are attributed to rhythmic changes in the activity of the enzyme N-acetyl transferase (NAT), an enzyme in the pathway for the synthesis of melatonin. A CR in the NAT activity was observed in isolated cultured pineal glands for 2 days by Binkley et al. (1978). Deguchi (1979a) was able to observe a CR for 3 days. If the cultured glands were not sampled for the first 2 days, a CR could be observed for the next 2 days (Kasai er al., 1979). Cells cultured from the pineal gland also exhibit a CR in NAT activity; this could be observed for 2 days but was damped out on the third day (Deguchi, 1979b). The difference between day and night-time activity appears to be the same for both cultured glands and for cells; there is a ten-fold increase at night compared to the daytime value. Isolated pineal tissue of the house sparrow when transplanted into the eye of an arrythmic host, restores the CR (Zimmerman and Menaker, 1979). The host was made arrhythmic by removing the pineal gland. The restored rhythm has the phase of the donor, evidence that the pineal acts as a pacemaker in this case. Some portions of the nervous system of insects have a pacemaker role for the CR. When these CNS areas are surgically isolated they can be cultured and then transplanted into other individual insects; by this procedure the peak of the circadian rhythm in the recipient insect is changed to that of the donor. This has been done with silk moths, Dros~~~j~ff and cockroaches (Takahashi and Zatz, 1982; Page, 1982). The adrenal gland is another organ in which CRs are observed under culture conditions. It was observed in cultured adrenal glands from hamster (Andrews and Folk, 1963) that the oxygen consumption varied from 60:{, above the mean at midnight to 60% below the mean at midday. The rate of uptake of [‘4Cfacetate and its incorporation into steroids was
RANJANA KADLE and G. EDGAR FOLK.
774
6-8 times greater at midnight than at midday (Andrews, 1968). A similar increase in the secretion of corticosterone by .cultured adrenal glands was observed (Shiotsuka et nl., 1974). When the hamster adrenals were cultured from animals conditioned to LD 12:12; the respiratory peaks of the glands were found during evening hours (Andrews, 1971). In contrast to the above findings, O’Hare and Hornsby (1975) observed no significant circadian variations in the corticosterone output of cells stimulated (by ACTH) or of unstimulated cells cultured from the rat adrenal cortex. This could be due to a desynchronization of cells or due to species difference between rat and hamster. However, further studies need to be conducted to substantiate this. A CR in the isolated rat heart has been reported by Tharp and Folk (1965). Isolated hearts, as well as isolated rat heart cells and heart cell networks showed a circadian and 12 hr pattern of rhythmic activity in the rate of contraction. Since the networks were formed from scattered isolated heart cells. the rhythmicity in the whole organ cannot be due to a particular arrangement of cells. Rather, it appears to reside in individual cells which are then synchronized. Cultured liver cells have also been shown to exhibit a CR (Hardeland, 1973a, b). Cultured liver cells from 3-7-day-old rat pups demonstrated a CR in tyrosine aminotransferase (TAT) activity when cultured under LD 12: 12 conditions. The highest activity in TAT was observed during the light period while the lowest was recorded in the dark period. When the cells were in constant light, this oscillation continued for up to 5 days; hence this activity is truly circadian.
ENVIRONMENTAL
CONDITIONS
AFFECTING
CR
The duration of illumination is known to have considerable influence on the daily activity of animals. The effect of continuous light on the daily activity of many animals is described by “Aschoffs rule”. This states that “in continuous light with increasing intensity of illumination, light-active animals increase their spontaneous frequency, while dark-active animals decrease it”. In continuous darkness the dark-active animal takes on its free-running rhythm. Thus, in continuous darkness or light the usual periodicity of the rhythm can either increase or decrease (from 24 to approx. 26 or 22 hr). Even in continuous darkness a single flash of light has been known to act as a “zeitgeher” or time-giver (Folk, 1974). Since CR are observed in whole animals as well as organ and cell cultures, it is plausible that the basis for the animal CR resides at the cellular level. Although it has been suggested that in cultured chick pineal glands the photosensitivity may reside at the enzymatic level (Wainwright and Wainwright, 1981) the exact mechanism by which various cultured cells or organs may perceive light is not yet clear. Thus it appears that it is important to consider the duration of illumination when working with cell cultures. Most of the cells in culture are subjected to constant darkness in the incubator, with occasional flashes of light due to opening of the incubator door. It is possible that these flashes are “xitgeher.s” to the cells. More information regarding this aspect is
JK
needed before reaching any conclusions, but the following experiments do support the concept. Different illuminating conditions affect the CR in some pineal cell cultures. A CR in the NAT activity, in cells kept in constant darkness, could be observed (Deguchi, 1979b) after the second day, up to two more days. However, the difference in the daytime and night-time activity damped out on the fifth day, the daytime levels being higher and the night-time levels being lower than the levels in cells cultured under LD 12: 12 conditions. This could be due to a desynchronization in the rhythm of activity of the individual cells. In other experiments, it was concluded that the pineal glands have a kind of “memory” of the timing of the lighting conditions to which their donors were exposed, since the occurrence of the peak of NAT activity depended on the time the chickens were killed (Binkley et ul., 1978). This suggests that it is important to consider the time at which the cells are being taken out of the incubator for further experiments. The CR in TAT activity in cultured liver cells is also affected by constant lighting conditions, it disappeared in approx. 2 weeks in constant light. The enzyme activity does not stabilize at the mean value of oscillation but rather near the minimum of the oscillatory state. Hardeland (1973a) suggests that this is due to the cells being desynchronized with respect to each other. The rhythmicity could be reinduced by just I hr period of darkness (Hardeland, 1973b). These observations further stress the importance of proper lighting conditions for cell cultures. The next question is whether cells in culture can entrain to reversed photoperiods. This proved to be the case with pineal cells in culture (Deguchi, 1979b). These observations indicate that although cellular CRs are endogenous, they may entrain to unusual lighting conditions.
EFFECT OF RNA AND PROTEIN INHIBITORS
SYNTHESIS
The effect of Actinomycin D, an RNA synthesis inhibitor, on CR has been studied. The daytime NAT activity in cultured pineal glands from rats could be stimulated by isoproterenol, but only after a lag period in which virtually no enzyme is synthesized (Romero and Axelrod, 1975). It has been suggested by Zatz et ul. (1976), on the basis of their experiments with Actinomycin D, that this lag period is the time required for RNA synthesis. Actinomycin D also abolishes secretory rhythms of corticosteroid output in the adrenal cultures (Andrews and Shiotsuka, 1970). These observations suggest that RNA synthesis is a necessary step in the generation of CR. It is, however, possible that Actinomycin D may have a direct effect on corticosteroid secretion, instead of its effect on CR. There is evidence to suggest that the CR may be regulated at the post transcriptional level as well (Hardeland and Stephan, 1974; Hardeland, 1976). A CR in TAT activity in the cultured rat liver cells could be only partially abolished by 5-azacytidine and a-amanitin (both acting at the transcriptional
Circadian rhythms in animal level). However, cordycepin, a drug acting posttranscriptionally could completely inhibit the increase in TAT activity in the light period. A quantitation of mRNA for TAT at different times is yet to be done. Nevertheless these studies do indicate the and postpresence of both transcriptional transcriptional components in the regulation of CR. Protein synthesis is also considered to be necessary for the expression of CR. The night-time activity of NAT could be blocked by cycloheximide (Deguchi, 1979a). Similar results have been obtained with puroand cycloheximide in the Aplysia eye; mycin Rothman and Strumwasser (1976) observed a phase shift in the rhythm of CAP, indicating a requirement for protein synthesis for the proper expression of CR. Again, the possibility of the effect of cycloheximide on the CR being due to its direct effect on the NAT levels or CAP instead of its effect on CR cannot be ruled out. The effect of these protein synthesis inhibitors on the CR should be considered when interpreting data on the effect of these agents on some cellular functions.
775
cell cultures
cadian rhythm in the culture must be understood taken into account.
and
CONCLUSIONS
Recent studies with cell and organ cultures have demonstrated that circadian rhythms do exist in the culture systems studied, suggesting that such rhythms might be a characteristic of many other culture systems. A definitive statement cannot be made, because there is a lack of data and not because of negative results. Further studies need to be carried out on possible circadian rhythms in all cell and organ cultures and on the standardization of conditions for their proper expression.
REFERENCES
Andrews R. V. (1968) Daily variation in membrane cultured
hamster
adrenals.
Camp.
Biochem.
flux of
Physiol.
26,
469. CELL CYCLES
AND CR
Studies on cell cycles of unicellular animals (Edmunds and Adams, 1981) as well as of cells cultured from higher animals (Rensing and Goedeke, 1976) suggest that cell cycles do exhibit CR. Human embryonic fibroblasts, rat liver and rat hepatoma cells show circadian variations in the percentage of cells in different stages of the cell cycle. However, no significant changes in the percentage of cells in different phases have been recorded in HeLa cells (Rensing et al., 1974). This is relevant to the interpretation of data on growth curves of cell cultures. Moreover, if cell cycles do exhibit circadian variations, this would be a simple means of synchronizing the cells in a particular stage of the cell cycle. Further, many investigators, e.g. Scheving et al. (1978), have suggested that this aspect could be taken advantage of in cancer chemotherapy. By knowing the rhythm of toxicity of an agent to the host cells the administration of a cancer drug can be timed so as to have a minimal effect on the host cells.
APPLICATIONS
The above account has established that isolated organs and cells can retain or develop a circadian physiological rhythm; this is contrary to the reasonable assumption made by many workers that the physiological peaks of individual cultured cells will not be synchronized. The above evidence also leads to the conclusion that flashes of light may change the circadian rhythm of cultured cells or organs. An example of a possible experimental error can be found in a hypothetical case of an investigator who wishes to add a pulse of hormone to the media around cultured cells. There may be only one period of the day when these cells are vulnerable to the hormone. Negative results may be assigned to a wrong explanation. If the cells are in darkness, the flash of light when the door is opened may change the time of day of vulnerability. Thus, a potential cir-
Andrews R. V. (1971) Circadian rhythms in adrenal organ cultures. Gegenhaurs morph. Jh. 117, 89. Andrews R. V. and Folk G. E. Jr. (1963) Circadian metabolic patterns
in cultured hamster adrenals. Camp. Bio11, 393. Andrews R. V. and Shiotsuka R. (1970) The effect of antinimycin D on the in z+tro adrenal secretory rhythm of the hamster. Comp. Biochem. Physiol. 36, 353. Binklev S. A.. Riebman J. B. and Reillv K. B. (1978) The pineal gland: a biological clock in ilitri. S&r&202, ‘I 198. Deguchi T. (I 979a) Circadian rhythm of serotonin N-acetyl transferase activity in organ culture of chicken pineal gland. Science 203, 1245. Deguchi T. (I 979b) A circadian oscillator in cultured cells of chicken pineal gland. Narure, Lord. 282, 94. Edmunds L.-N.. Jr. and Adams K. J. (1981) Clocked cell cycle clocks. Science 211, 1002-1013. Eskin A., Corrent G., Lin C. Y. and McAdoo D. J. (1982) Mechanism for shifting the phase of a circadian rhythm by serotonin: involvement of CAMP. Proc,. rum. Acad. them.
Physiol.
Sci. U.S.A.
79, 660.
Folk G. E., Jr. (1974) In Textbook qf Emironmentd PhJ,sio/og_v, p. 24. Lea and Febiger, Philadelphia. Hardeland R. (1973a) Circadian rhythmicity in cultured liver cells. I. Rhythms in tyrosine aminotransferase activity and inducibility and in [ZH]leucine incorporation. Int. J. Biochem.
4, 581.
Hardeland R. (1973b) Circadian rhythmicity in cultured liver cells. 11. Reinduction of rhythmicity in tyrosine aminotransferase activity. Ini. J. Biochem. 4, 591. Hardeland R. (1976) Further evidence for a posttranscriptional component in the regulation of circadian rhythmicity in cultured liver cells. Possible significance of RNA processing. J. Inierdiscipl. Cycle Res. 7(4). 291. Hardeland R. and Stephan E. (I 974) Diurnal rhythms and post-transcriptional regulation of hepatic thyrosine aminotransferase and tryptophan oxygenase. J. Iniw&wipl. Cycle Res. 5(34), 247. Jacklet J. W. (1969) Circadian rhythm of optic nerve impulses recorded in darkness from isolated eye of Aplysia. Science 164, 562. Jacklet J. W. (1974) The effect of constant light and light pulses on the circadian rhythm in the eye of A&sI’u. J. Comp. Ph_vsiol. 90, 33. Kasal C. A., Menaker M. and Regino Perez-Polo J. (1979) Circadian clock in culture: N-acetyl transferase activity ot chick pineal glands oscillates in dro. Science 203, 656.
RANJANA KADLE and G. EDGAR FOLK. JK
176
O’Hare M. J. and Hornsby P. J. (1975) Absence of circadian rhythm of corticosterone secretion in monolayer cultures of adult rat adrenocortical cells. E.r~erientiu 31, 378. Page T. L. (1982) Transplantation of the cockroach circadian pacemaker. Science 216, 73-75. Rcnsing L., Goedeke K., Wassmann G. and Broich G. (I 974) Presence and absence of daily rhythms of nuclear size and DNA syntheses of different normal and transformed cells in culture. J. Interrliscipl. C,& Rm. 5(3-4). 267. Rensing L. and Goedeke K. (1976) Circadian rhythm and cell cycle: possible entraining mechanisms. Chronohiologiu 3, 53.
Romero J. A. and Axelrod J. (1975) Regulation and sensitivity to /I-adrenergic stimulation in induction of pincal N-acetyl transferase. Proc. nutn. Acud. Sci. L:.S.A. 72(5). 1661.
Rothman B. S. and Strumwasser F. (I 976) Phase shifting of the circadian rhythm of neuronal activity in the isolated Aplysiu eye with puromycin and cycloheximide. J. gen. Physiol.
Scheving
68, 359.
L. E.. Burns
E. R., Pauly
J. E. and Tsai T. H.
(1978) Circadian rhythm variation m cell division of the mouse alimetary tract, bone marrow and cornea1 epithelium. Anur. Rec. 191(4), 479. Shiotsuka R.. Jovonovich J. and Jovonovtch J. A. (1974) Circadian and ultradian rhythms in adrenal organ cultures, C‘hronohiolo~icr l(Suppl. I ), 109. Takahashi J. S. and Zatz M. (I 982) Regulatton of circadian rhythmicity. Science 217, 1104. Tharp G. D. and Folk G. E. Jr. (1965) Rhythmic changes in rate of the mammalian heart and heart cells during prolonged Isolation. C‘onzp. Bioch~~nr. Ph~~.sid. 14, 255. Wainwright S. D. and Wainwright L.K. (1981) The rclationship between variations in levels of serotonm acetyltransferase activity to CAMP content m cultured chick pineal glands. Cm. J. Biochem. 59(8). 593-601. Zatz M.. Romero J. A. and Axelrod J. (1976) Diurnal variation in the requirement for RNA synthesis in the induction of h’-acetyl transferase. Biochrrn. Phormuc~. 25, 903. Zimmerman N. H. and Menaker M. (1979) The pmeal gland: a pacemaker within the circadian system of the house sparrow. Proc. natn. Acud. Sci. U.S.A. 76, 999.
Printedin Great Biitain
c
030%9629/83 $3.00 + 0.00 1983 Pergamon Press Ltd
MINIREVIEW
IMPORTANCE OF CIRCADIAN RHYTHMS ANIMAL CELL CULTURES
IN
RANJANA KADLE and C. EDGAR FOLK, JR. Department
of Physiology
and Biophysics, The University of Iowa, Telephone (3 19) 353-4152
Iowa City. IA 52242, USA,
Abstract-l. Circadian rhythms are a characteristic feature of many cell and organ cultures. 2. Such rhythms may be important in the interpretation of data from cells in culture. 3. More examples of circadian rhythms in tissue culture are badly needed to understand phenomena. However, they will only be expressed under optimum and well-understood conditions.
INTRODUCTlON
The importance of circadian rhythms (CR) in cellular function is sometimes overlooked when working with culture systems. Much of the data from such systems is interpreted without considering the possibility of some effect being due to circadian variations. The presence of CR is a specific characteristic of whole organisms (unicellular as well as multicellular). Although the observations made on unicellular organisms cannot be extended easily to isolated cells derived from more complex multicellular animals, it is plausible that the basic unit of the circadian clock resides in these isolated individual cells. A number of studies have been conducted to determine whether CR are exhibited by cells or organs when isolated from the body. In this paper we review some of these results, and we stress the importance of exploring for the presence of such rhythms whenever culture systems are used. EVIDENCE FOR THE PRESENCE OF CR IN ORGAN AND CELL CULTURES
There is evidence for CR in cultures of: (1) the eye of A&&, (2) pineal glands and cells cultured from these glands. both avian and mammalian, (3) nervous tissue of insects, (4) adrenal glands and cells cultured from these, (5) isolated heart and heart cell networks and (6) liver cells. The isolated eye of the sea hare, Apl_vsia culifornicu, displays CR of large amplitude in the compound action potentials (CAP) of the optic nerve impulses (Jacklet, 1969: Eskin et ul., 1982). These can be recorded Sor up to 2 weeks in constant darkness (Jacklet. 1974). The peak of the CAP activity, in constant darkness, for animals entrained to a LD 12: I2 cycle is at the projected dawn, while the minimum is at the projected dusk (Rothman and Strumwasser, 1976). In constant darkness the correspondence of the activity cycles to the cycle to which the animals were entrained decreased gradually. They acquired a free-running rhythm. 773
this
The cultured chick pineal gland displays a CR in the secretion of the hormone melatonin. Maximum levels are observed during night-time and the minimum during daytime. The variation in the levels of melatonin are attributed to rhythmic changes in the activity of the enzyme N-acetyl transferase (NAT), an enzyme in the pathway for the synthesis of melatonin. A CR in the NAT activity was observed in isolated cultured pineal glands for 2 days by Binkley et al. (1978). Deguchi (1979a) was able to observe a CR for 3 days. If the cultured glands were not sampled for the first 2 days, a CR could be observed for the next 2 days (Kasai er al., 1979). Cells cultured from the pineal gland also exhibit a CR in NAT activity; this could be observed for 2 days but was damped out on the third day (Deguchi, 1979b). The difference between day and night-time activity appears to be the same for both cultured glands and for cells; there is a ten-fold increase at night compared to the daytime value. Isolated pineal tissue of the house sparrow when transplanted into the eye of an arrythmic host, restores the CR (Zimmerman and Menaker, 1979). The host was made arrhythmic by removing the pineal gland. The restored rhythm has the phase of the donor, evidence that the pineal acts as a pacemaker in this case. Some portions of the nervous system of insects have a pacemaker role for the CR. When these CNS areas are surgically isolated they can be cultured and then transplanted into other individual insects; by this procedure the peak of the circadian rhythm in the recipient insect is changed to that of the donor. This has been done with silk moths, Dros~~~j~ff and cockroaches (Takahashi and Zatz, 1982; Page, 1982). The adrenal gland is another organ in which CRs are observed under culture conditions. It was observed in cultured adrenal glands from hamster (Andrews and Folk, 1963) that the oxygen consumption varied from 60:{, above the mean at midnight to 60% below the mean at midday. The rate of uptake of [‘4Cfacetate and its incorporation into steroids was
RANJANA KADLE and G. EDGAR FOLK.
774
6-8 times greater at midnight than at midday (Andrews, 1968). A similar increase in the secretion of corticosterone by .cultured adrenal glands was observed (Shiotsuka et nl., 1974). When the hamster adrenals were cultured from animals conditioned to LD 12:12; the respiratory peaks of the glands were found during evening hours (Andrews, 1971). In contrast to the above findings, O’Hare and Hornsby (1975) observed no significant circadian variations in the corticosterone output of cells stimulated (by ACTH) or of unstimulated cells cultured from the rat adrenal cortex. This could be due to a desynchronization of cells or due to species difference between rat and hamster. However, further studies need to be conducted to substantiate this. A CR in the isolated rat heart has been reported by Tharp and Folk (1965). Isolated hearts, as well as isolated rat heart cells and heart cell networks showed a circadian and 12 hr pattern of rhythmic activity in the rate of contraction. Since the networks were formed from scattered isolated heart cells. the rhythmicity in the whole organ cannot be due to a particular arrangement of cells. Rather, it appears to reside in individual cells which are then synchronized. Cultured liver cells have also been shown to exhibit a CR (Hardeland, 1973a, b). Cultured liver cells from 3-7-day-old rat pups demonstrated a CR in tyrosine aminotransferase (TAT) activity when cultured under LD 12: 12 conditions. The highest activity in TAT was observed during the light period while the lowest was recorded in the dark period. When the cells were in constant light, this oscillation continued for up to 5 days; hence this activity is truly circadian.
ENVIRONMENTAL
CONDITIONS
AFFECTING
CR
The duration of illumination is known to have considerable influence on the daily activity of animals. The effect of continuous light on the daily activity of many animals is described by “Aschoffs rule”. This states that “in continuous light with increasing intensity of illumination, light-active animals increase their spontaneous frequency, while dark-active animals decrease it”. In continuous darkness the dark-active animal takes on its free-running rhythm. Thus, in continuous darkness or light the usual periodicity of the rhythm can either increase or decrease (from 24 to approx. 26 or 22 hr). Even in continuous darkness a single flash of light has been known to act as a “zeitgeher” or time-giver (Folk, 1974). Since CR are observed in whole animals as well as organ and cell cultures, it is plausible that the basis for the animal CR resides at the cellular level. Although it has been suggested that in cultured chick pineal glands the photosensitivity may reside at the enzymatic level (Wainwright and Wainwright, 1981) the exact mechanism by which various cultured cells or organs may perceive light is not yet clear. Thus it appears that it is important to consider the duration of illumination when working with cell cultures. Most of the cells in culture are subjected to constant darkness in the incubator, with occasional flashes of light due to opening of the incubator door. It is possible that these flashes are “xitgeher.s” to the cells. More information regarding this aspect is
JK
needed before reaching any conclusions, but the following experiments do support the concept. Different illuminating conditions affect the CR in some pineal cell cultures. A CR in the NAT activity, in cells kept in constant darkness, could be observed (Deguchi, 1979b) after the second day, up to two more days. However, the difference in the daytime and night-time activity damped out on the fifth day, the daytime levels being higher and the night-time levels being lower than the levels in cells cultured under LD 12: 12 conditions. This could be due to a desynchronization in the rhythm of activity of the individual cells. In other experiments, it was concluded that the pineal glands have a kind of “memory” of the timing of the lighting conditions to which their donors were exposed, since the occurrence of the peak of NAT activity depended on the time the chickens were killed (Binkley et ul., 1978). This suggests that it is important to consider the time at which the cells are being taken out of the incubator for further experiments. The CR in TAT activity in cultured liver cells is also affected by constant lighting conditions, it disappeared in approx. 2 weeks in constant light. The enzyme activity does not stabilize at the mean value of oscillation but rather near the minimum of the oscillatory state. Hardeland (1973a) suggests that this is due to the cells being desynchronized with respect to each other. The rhythmicity could be reinduced by just I hr period of darkness (Hardeland, 1973b). These observations further stress the importance of proper lighting conditions for cell cultures. The next question is whether cells in culture can entrain to reversed photoperiods. This proved to be the case with pineal cells in culture (Deguchi, 1979b). These observations indicate that although cellular CRs are endogenous, they may entrain to unusual lighting conditions.
EFFECT OF RNA AND PROTEIN INHIBITORS
SYNTHESIS
The effect of Actinomycin D, an RNA synthesis inhibitor, on CR has been studied. The daytime NAT activity in cultured pineal glands from rats could be stimulated by isoproterenol, but only after a lag period in which virtually no enzyme is synthesized (Romero and Axelrod, 1975). It has been suggested by Zatz et ul. (1976), on the basis of their experiments with Actinomycin D, that this lag period is the time required for RNA synthesis. Actinomycin D also abolishes secretory rhythms of corticosteroid output in the adrenal cultures (Andrews and Shiotsuka, 1970). These observations suggest that RNA synthesis is a necessary step in the generation of CR. It is, however, possible that Actinomycin D may have a direct effect on corticosteroid secretion, instead of its effect on CR. There is evidence to suggest that the CR may be regulated at the post transcriptional level as well (Hardeland and Stephan, 1974; Hardeland, 1976). A CR in TAT activity in the cultured rat liver cells could be only partially abolished by 5-azacytidine and a-amanitin (both acting at the transcriptional
Circadian rhythms in animal level). However, cordycepin, a drug acting posttranscriptionally could completely inhibit the increase in TAT activity in the light period. A quantitation of mRNA for TAT at different times is yet to be done. Nevertheless these studies do indicate the and postpresence of both transcriptional transcriptional components in the regulation of CR. Protein synthesis is also considered to be necessary for the expression of CR. The night-time activity of NAT could be blocked by cycloheximide (Deguchi, 1979a). Similar results have been obtained with puroand cycloheximide in the Aplysia eye; mycin Rothman and Strumwasser (1976) observed a phase shift in the rhythm of CAP, indicating a requirement for protein synthesis for the proper expression of CR. Again, the possibility of the effect of cycloheximide on the CR being due to its direct effect on the NAT levels or CAP instead of its effect on CR cannot be ruled out. The effect of these protein synthesis inhibitors on the CR should be considered when interpreting data on the effect of these agents on some cellular functions.
775
cell cultures
cadian rhythm in the culture must be understood taken into account.
and
CONCLUSIONS
Recent studies with cell and organ cultures have demonstrated that circadian rhythms do exist in the culture systems studied, suggesting that such rhythms might be a characteristic of many other culture systems. A definitive statement cannot be made, because there is a lack of data and not because of negative results. Further studies need to be carried out on possible circadian rhythms in all cell and organ cultures and on the standardization of conditions for their proper expression.
REFERENCES
Andrews R. V. (1968) Daily variation in membrane cultured
hamster
adrenals.
Camp.
Biochem.
flux of
Physiol.
26,
469. CELL CYCLES
AND CR
Studies on cell cycles of unicellular animals (Edmunds and Adams, 1981) as well as of cells cultured from higher animals (Rensing and Goedeke, 1976) suggest that cell cycles do exhibit CR. Human embryonic fibroblasts, rat liver and rat hepatoma cells show circadian variations in the percentage of cells in different stages of the cell cycle. However, no significant changes in the percentage of cells in different phases have been recorded in HeLa cells (Rensing et al., 1974). This is relevant to the interpretation of data on growth curves of cell cultures. Moreover, if cell cycles do exhibit circadian variations, this would be a simple means of synchronizing the cells in a particular stage of the cell cycle. Further, many investigators, e.g. Scheving et al. (1978), have suggested that this aspect could be taken advantage of in cancer chemotherapy. By knowing the rhythm of toxicity of an agent to the host cells the administration of a cancer drug can be timed so as to have a minimal effect on the host cells.
APPLICATIONS
The above account has established that isolated organs and cells can retain or develop a circadian physiological rhythm; this is contrary to the reasonable assumption made by many workers that the physiological peaks of individual cultured cells will not be synchronized. The above evidence also leads to the conclusion that flashes of light may change the circadian rhythm of cultured cells or organs. An example of a possible experimental error can be found in a hypothetical case of an investigator who wishes to add a pulse of hormone to the media around cultured cells. There may be only one period of the day when these cells are vulnerable to the hormone. Negative results may be assigned to a wrong explanation. If the cells are in darkness, the flash of light when the door is opened may change the time of day of vulnerability. Thus, a potential cir-
Andrews R. V. (1971) Circadian rhythms in adrenal organ cultures. Gegenhaurs morph. Jh. 117, 89. Andrews R. V. and Folk G. E. Jr. (1963) Circadian metabolic patterns
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