Cell proliferation by cell cycle analysis in young and old dietary restricted mice

Mechanisms of Ageing and Development, 68 (1993) 151- 162

151

Elsevier ScientificPublishers Ireland Ltd.

CELL PROLIFERATION BY CELL CYCLE ANALYSIS IN YOUNG AND OLD DIETARY RESTRICTED MICE

M I N G H. LU, WILLIAM G. HINSON, A N G E L O T U R T U R R O , WlNSLOW G. SHELDON and R O N A L D W. H A R T Food and Drug Administration National Centerfor Toxicological Research ( NCTRJ, Jefferson, Arkansas 72079 (USA)

(Received October 30th, 1992)

SUMMARY The effect of dietary restriction (DR) on cell proliferation determined by cell cycle analysis in tissues of young and old mice was investigated. Using the percentage of S-phase cells as an index of cell proliferation, we found that DR inhibited cell proliferation in spleen and thymus in young mice. No significant changes were found in bone marrow and kidney in the ad libitum (AL) or DR mice regardless of age. In old mice, the DR effect was observed in spleen only. When age increased, a parallel decline in cell proliferation was evidenced by a reduced % of S-phase cells. DR produces a greater cell cycle effect in the young mice than in the old mice. The present data suggests that inhibition of cell proliferation by DR may be affected by type of tissue, age, length of DR, and capacity or rate of cell proliferation.

Key words." Cell proliferation; Cell cycle analysis; Dietary restriction; Aging; Mouse

INTRODUCTION Dietary restriction has been reported to extend maximum life span, retard onset of senescence, and lower incidences of naturally occurring and chemically-induced tumors in rodents [1-8]. DR may lengthen the time to the occurrence of various ageassociated diseases, including cancer [9-14]. Therefore, DR is useful as a modulator of various forms of chemical toxicity. While some of its effects have been known for over forty years, its mode of action still remains unclear. Cell proliferation appears Correspondence to." Ming H. Lu, Food and Drug Administration, National Center for ToxicologicalResearch (NCTR), Jefferson,Arkansas 72079, USA.

0047-6374/93/$06.00 © 1993 ElsevierScientificPublishers Ireland Ltd. Printed and Published in Ireland

152

to be an important biomarker since DR may inhibit the growth of normal cells and/or cell variants that have the capacity to become neoplasm [9]. Holehan and Merry reported that DR inhibited synthesis of DNA in a number of tissues [15]. DR inhibits cell division in specific tissue in rats [16] and mice [17,18]. Cell proliferation rate can be estimated by analyzing the cell cycle of tissues by flow cytometry [19]. Cell cycle analysis performed by flow cytometry [20] is used to measure the fraction of cells in various phases of the cell cycle based on their DNA content. The data obtained from analysis of DNA distributions in each phase of the cell cycle provides information about the proliferative activity. The percentage of cells in the S phase of the cell cycle can be defined as an index of proliferation [21,221. In a previous article, we examined the effect of DR on cell proliferation in the tissues of DR rats by cell cycle analysis using flow cytometry [19,20]. The data indicated that in rats, the tissues from old animals were less sensitive to DR when compared to young animals [23,24]. In the present study, we examined the effect of DR on cell proliferation by cell cycle analysis in thymus, spleen, bone marrow, and kidney tissues of male mice. Also, we compared the effect of DR on cell cycle analysis of these tissues between young (5-month-old) and old (30-month-old) mice. MATERIALS AND METHODS

Animals and feeding regimens Male B6C3FI mice obtained from the N C T R breeding colony were used. The housing of animals and feeding regimens have previously been described [24]. The animals were raised at 23°C and 50% relative humidity in a specific pathogen free (SPF) facility and were maintained on a 12-h light-dark cycle with the light on from 0600 to 1800 h daily. The mice were housed singly in plastic cages with hardwood chip bedding and metal tops. All mice were fed AL a standard NIH-31 diet at 1100 h daily throughout 13 weeks of age. At 14 weeks of age, the mice were divided into two groups: a control group which received feed pellets AL and a DR group which was started on a restricted diet. Initially, the DR group received 90% of the AL consumption for 1 week, 75% of the AL consumption during the second week (15th week of age), and 60% of the control consumption starting at 16 weeks of age until at 5 months (1 month post-initiation of DR for young mice) or 30 months (26 months post-initiation of DR for old mice) of age at which time the mice were sacrificed. The control mice for both young and old groups were similarly sacrificed at 5 or 30 months of age. Five to six mice were used in each group as shown in Tables 1 and 2. Mice from the DR group received supplemental vitamins to maintain the same level of vitamins as control mice. All mice were provided water ad libitum. One month before sacrifice, the photoperiod was shifted by 12 h to synchronize the feeding times (at 1100 h) so the DR mice ate at about the same time period during which the AL animals fed most actively within the light-dark cycle.

153

Preparation of tissues At 5 or 30 months of age, mice were killed (between 1300 and 1400 h) by asphyxiation using carbon dioxide. The bone marrow tissue was collected from the cleaned femur and tibia by flushing the bone with a 21-gauge needle [24] attached to a 3-ml syringe filled with buffer solution that contains the mixture of citrate-sucrosedimethylsulfoxide [25]. Thymus was removed from the thoracic cavity; kidney and spleen tissues were removed from the abdominal cavity. They were then sliced into 1-mm thick sections for complete immersion of tissue in the buffer solution. All tissues were collected separately in plastic vials (14 mm x 50 mm, Walter Sarstedt, Inc., Princeton, NJ) containing 200 #1 of buffer solution [25] and immediately frozen in liquid nitrogen until all animals were sacrificed. They were then stored in a -80°C freezer until assayed.

Flow cytometric cell cycle analysis Measurement of DNA content is the basis of cell cycle analysis by flow cytometry. The modified method of Vindelov et al. [25] was used for staining nuclei. Nuclear suspension of the four tissues was made from frozen tissues as described previously [24]. Nuclei were isolated by trypsinization of the tissue suspension. The nuclear DNA was stained with propidium iodide (PI) (Calbiochem-Behring Corp., La Jolla, CA). The prepared samples were kept at 4°C and analyzed on the flow cytometer within 60 min of addition of the PI solution. Cellular DNA content was measured by a FACScan Flow Cytometer (Becton Dickinson, San Jose, CA) equipped with an Argon-laser emitting at 488 nm to excite the DNA-associated PI to fluoresce at a wavelength of 630 nm. Histograms of 10 000 cells were recorded for each sample at a flow rate of 100-200 cells/s. Data collection was done using the Consort 30 Data Acquisition Program (Becton Dickinson) and an HP310 Computer (Hewlett Packard, Palo Alto, CA). Analysis of the DNA histogram was performed using Becton Dickinson's DNA/Cell Cycle Analysis Software, Revision B, based on the SFIT polynomial model [26]. Populations of nuclei in the various cell cycle phases (based on DNA content) were assumed to represent the cell populations and were expressed as percentage of the entire population of the histogram obtained. Cell proliferation in this study is expressed in terms of the total percentage of S-phase cells [27].

Methodology for leukocyte identification and for nephropathy and glomerulosclerosis assessment Leukocyte counting was performed by using the automated counting equipment, the Coulter Counter $70 (Coulter $70 Reference Manual, 1980; Coulter Electronics, Inc., Hialeah, FL). Nephropathy and glomerulosclerosis were graded by the methods reported by Coleman et al. [28]. Both types of lesions were graded on a scale from 0 to 4 with 0 representing normal, 1 minimal, 2 mild, 3 moderate, and 4 severe.

154

Statistical analysis All samples were run in duplicate to obtain the mean. All numerical data in the treatment groups were expressed as mean 4- standard error of mean (S.E.M.). Statistical comparison of flow cytometric data was done using the unpaired Student's t-test. A P-value of < 0.05 was considered a statistically significant difference. All the statistics were performed using the two-tailed tests. RESULTS

Table I summarizes the cell cycle analysis data derived from thymus, spleen, bone marrow, and kidney of young and old male mice of the AL and DR groups. Cytometric analysis of the tissues from the AL and DR animals indicated some variations in the cell cycle components. The data showed that even with the large differences obtained in body weights, the differences in cell cycle distributions seemed to be tissue/organ dependent. Similar to our previous findings in the rat, all tissues showed that the G0/Grphase of the cell cycle exhibited the largest percentage of TABLE I CELL CYCLE ANALYSIS OF TISSUES IN MALE EACH CELL CYCLE PHASE (MEAN ± S.E.M.)

Tissue

B6C3F I MICE. PERCENT

Control (ad libitum)

O F C E L L S II31

Dietary Restricted

Young (n = 5)

Old (n = 6)

Young (n = 6)

Old (n = 5)

79.00 ± 1.38 a,* 11.90 ± 0.92 b'** 9.10 ± 1.20c'i"

86.05 ± 2.44 d'* 8.58 ± 1.88"* 4.92 ± 1.20"~

85.33 ± 5.23 a 9.00 + 3.06 b 5.67 ± 2.21 c

89.50 ± 2.30 d 6.70 ± 1.69 3.90 ± 1.24

81.30 ± 1.50 a 13.20 ± 2.42 e 5.70 ± 1.29"*

79.08 ± 2.94 b 12.67 ± 2.78 d 8.25 + 0.63**

85.38 ± 1.02 a'* 9.29 ± 2.62 c 6.21 ~ 0.68 t

83.0 ± 1.76 b'* 8.3 ± 1.75 d 8.5 ± 0.71"[

63.50 ± 1.14" 28.90 ± 1 . 3 2 ' * 7.70 ± 0 . 6 0 c ' t

76.67 ± 1.34" 17.00 ± 0.82** 6.50 ± 1.19"["

64.58 -4- 2.65 a 29.79 ± 2.70 b 5.58 ± 1.40 c

78.10 ± 2.71 a 16.10 ± 2.46 b 5.90 ± 0.97

91.60 ± 0.58* 4.20 ± 0.40** 4.10 ± 0.37

85.00 ± 1.38" 10.33 + 2.39** 4.50 ± 1.04

92.25 :t: 0.76 a 4.08 ± 0.67 b 3.75 ± 0.25 c

86.00 .4- 0.95 a 9.60 + 1.20 b 4.60 ± 0.58 c

Thymus Gl S G2M

Spleen G1 S G2M

Bone marrow Gl S G2M

Kidney G1 S G2M

Figures with at least one similar superscript in each tissue are different significantly ( P < 0.05). number of animals used; G1, P r e - D N A synthesis period; S, D N A synthesis period; G 2 M , p o s t - D N A synthesis period and mitosis period.

n,

155 cells, the S-phase cell population was intermediate, the G2M-phase showed the least. Bone marrow exhibited the highest proliferative activity with the percentage of cells in the S-phase being greater than that observed in the other tissues. This observation was consistent both in the AL and DR groups.

Body weight changes The body weights (g) expressed in mean 4- S.E.M. of 5-month-old AL and DR mice on sacrifice day were 32.40 4- 1.15 and 27.65 ± 0.58, respectively. This represents approximately 15% reduction in body weight due to DR occurring for 1 month. The mean body weights of 30-month-old AL and DR mice on the day of sacrifice were 38.53 4- 1.01 and 30.74 4- 0.84, respectively. This indicates about 20% body weight reduction caused by the long-term DR in old animals. These differences between the AL and DR mice were statistically significant (P <0.05) both in the young and old groups.

Effects of dietary restriction and age In thymus, DR affected significantly the distribution of cells in all phases of the cell cycle in young mice (Table I). The percentage of G 1 cells, perhaps Go cells proposed by Lajtha [29], increased in the DR group when compared to the AL group (79.0% vs. 85.3%). Also, the percentage of S-phase cells in young DR mice were significantly reduced when compared to young AL controls (11.9% vs. 9.0%). The percentage of cells in the G2M-phase in young DR mice were reduced significantly (P < 0.05) when compared to young AL controls (9.1% vs. 5.7%). Though the percentage of cells in S- and G2M-phases in the old animals tended to decrease due to DR, the differences between AL and DR groups (Table I) were not significant. The percentage of G0/G1 cells increased significantly in the old DR mice (Table I). The percentage of G0/Gl cells in the young mice was significantly lower than in old mice in the AL group (79.0 vs. 86.1). Though a tendency for G0/G1 cells to increase in old mice was seen in the DR group, the difference was not significant. The percentage of cell population in S-and G2M-phases in young mice was always higher than those in old mice both in the AL and DR groups (Table I). In the spleen, the percentage of the G0/G1 cells were significantly (P < 0.05) increased in both young and old DR mice. Values for the G0/G1 cells of the AL and DR were 81.3% vs. 85.4% in the young, and 79.1% vs. 83.0% in the old (Table I). However, the percentage of G0/GI cells in old DR mice decreased significantly when compared to young DR mice. The percentage of S-phase cells were found to be significantly decreased by DR in both the young and old mice (P <0.05). The percentage of S-cells remain unchanged between the old and young mice in both AL and DR groups. The G2M cells in old mice were significantly increased regardless of diet treatment (Table I). In bone marrow, no differences resulted from DR were observed in the G0/Gl and S phase cells in both young and old mice (Table I). No changes in the G2M

156 cells in old mice were found between the AL and DR groups. However, the differences in the G2M cells between AL and D R groups in young mice were statistically significant (P < 0.05). The percentage of G0/G1 cells in the old mice were significantly higher (P < 0.05) than those of the young mice in both the AL and DR groups. The percentage of S-cells were significantly lower in old mice than in young mice in both AL and D R groups. The percentage of G2M cells were comparable between young and old mice in the DR group. However, the percentage of the G2 M cells in old mice in the AL group was significantly lower than that of the young mice (P < 0.05) (Table I). In kidney tissue (Table I), DR did not cause any changes in the S-phase cells in either young or old mice. The G0/G1 and G2M cells also were not altered by the D R in the young or old mice. A significant decrease with age in G0/Gl cells occurred in both the AL and DR groups (P < 0.05). The percentage of G2M cells in young and old mice was comparable in A L group. However, the percentage of G2M cells in old mice of DR groups was significantly increased. Table II shows the leukocyte counts and the incidence of glomerulosclerosis, nephropathy, and lymphoma occurring in renal tissue in the 30-month-old male B6C3F l mice in the N C T R colony. Lymphoma is a multiple site occurring lesion. It occurred in 3/15 of kidneys of 30-month-old AL male mice vs. 0/15 in DR male mice. Nephropathy was found to occur more frequently in the AL than DR groups with low severity (Table II). Glomerulosclerosis in this strain of mice was found in all (100%) in 30-month-old males in both A L and D R groups. The lesions were found to be more severe in the AL mice than in the DR mice. The total leukocyte counts in DR mice were significantly lower than in their AL cohorts.

TABLE I1 LEUKOCYTE COUNTS AND INCIDENCESOF GLOMERULOSCLEROSIS,NEPHROPATHY, AND LYMPHOMAOCCURRED IN RENAL TISSUE OF 30-MONTH-OLDMALE B6C3FI MICE (N = 15)

AL DR

Leukocyte

GlomerulosclerosisNephropathy

Lymphomain kidney

8.17 ± 0.53 2.10 a- 0.11

15/15 (2.6) 15/15 (1.9)

3/15 0/15

11/15 (1) 4/15 (1)

AL, ad libitum group; DR, dietary restricted group. Figures in parenthesis denote lesion severity with 1 being the lowest and 4 being the highest rank. The leukocyte counts were given in mean cells x 103/mm 3 of blood + S.E.M.

157

DISCUSSION

We reported previously the effect of DR on distributions of cells within the cell cycle of rat tissues [24]. To our knowledge, the present study is the first on the effects of DR on cell cycle distribution within the cell cycle of thymus, spleen, bone marrow, and kidney tissues of mice. The differences of the body weights between AL and DR groups obviously reflect a significant effect of DR. The results of Lok et al. [17] also demonstrated that by reducing carbohydrates, a 40% DR in mice resulted in a significant decrease in body weight. According to the proliferative index defined in the Introduction and Materials and Methods sections, the decreased values of S-cells in thymus and spleen of DR animals may reflect a decrease in cell proliferative activity. Our data on the reduced G2M cells of thymus and bone marrow indicate that young animals are more susceptible to DR than old animals in same tissues. This finding is in agreement with our previous published data in which young DR rats also were more susceptible [24]. Winick and Noble [30] reported that if DR started early in development, it decreased hyperplastic growth of most organs and resulted in a lowered number of cells. Thus, the DR effect on cell proliferation appears to be age dependent. It is interesting to note that the stimulation effect of DR on the percentage of S-phase cells of bone marrow observed in young DR rats [241 was not seen in young DR mice. In thymus and spleen, G0/G1 cells in both young and old mice increased as a function of DR. However, the G0/GI Cells in bone marrow and kidney were not altered by DR. Thus, the effects of DR are tissue-dependent. Similar findings with this aspect was reported that DR had a greater effect on the mammary tissue than on other tissue [181. The increased percentage of G0/Gl cells might reflect an increased duration of Gl-phase in thymus and spleen which in turn may indicate an enhanced opportunity for DNA repair with DR, with all other factors remaining equal. An increase of G0/GI cells participating in DNA repair, as opposed to having fewer Sor S+G2M-cells involved in the fixation of genetic damages, could be regarded as one of the potential effects of DR to enhance DNA repair [31,32]. Cellular proliferation affects DNA adduct formation [33], fixation [34], promotion [35], and progression [36] of carcinogenesis. Cell division per se increases the risk of compounding various genetic errors, hence cells which are replicating possess increased susceptibility to mutagenesis and carcinogenesis [34]. Cell proliferation is an essential requirement of carcinogenesis [37,38]. A decrease in the rate of cell proliferation will reduce the fixation of DNA damage. An increase in the cell cycle time, particularly the length of Gl phase, will provide more time for DNA repair. Hence, if DR causes an enhanced DNA repair response as a result of a decrease in cell proliferation rate, it may also be expected to lower tumor incidence or lengthen the time to tumor occurrence. Many endogenous and exogenous factors can damage DNA in vivo, and DNA damage has been found to be associated with aging [39,40] and carcinogenesis

158

[40,41]. Loss of cell proliferative vigor and regression of tissues with a concomitant cell loss are indicators of the aging process [42]. Therefore, cell proliferation and DNA repair are related to aging and carcinogenesis [41,43]. Increases in rates of cell proliferation decreases the time to perform DNA repair [44]. Thus, if cells enter the proliferating cycle, the probability of promutagenic lesions in DNA (to remain unrepaired before DNA synthesis) ensues with increased risk of base-mispairing.and mutations. If DR slows down cell proliferation, it might lengthen the duration of the G0/Gl phase during which damaged cells would have a longer time for DNA repair. The immune function in animals declines with age [45,46]. The maintenance of the immune system requires constant normal cell proliferation in specific tissues. A higher percentage of S-cells reflects the higher probability of a strong immune capacity. The differences in percentage of S-cells in thymus and bone marrow between the young and old mice suggest that there is a possibility that young adult mice might have more immune activity than old mice. This phenomenon was also observed in our previous study in male Fischer 344 rats [24]. However, a portion of S-phase cells in young adult mice, if not become fully mature, might be immature progenitor cells lacking effector function. Similar to the rat study [24], both AL and DR mice maintained an order of distributions of the percentage of cells in various phases of cell cycle: G0/G~ > S > G2M. Also, chronic DR apparently did not alter functions of these tissues. However, in thymus and spleen DR appears to lengthen Grphase preferentially over other phases of the cell cycle. All four tissues examined in this study with the exception of kidney are involved in the immune system. The immune system of rodents has been reported to be strongly influenced by DR [47]. In thymus, it has been reported that 10 weeks of DR in mice if started early at 1 month of age lowered the mitotic index by 20%. When DR was started late at 8 months of age the mitotic rate did not change [48]. Our mice were started on DR at 16 weeks of age, the loss of the S-cells avei'aged 23% for both young and old mice. Weindruch and Walford [47] reported that in mice thymocyte replication and numbers decreased with DR. They claimed that perhaps DR retarded losses of some types of thymic epithelial cells. Our data shows that the percentage of S-cells in thymus decreased with DR. Whether or not the reduction of S-cells involves thymosin hormone producing cells is not known. In rats, DR started at early growth stage would result in decreased cell numbers [29]. In spleen, it was reported that DR decreased the number of nucleated cells per spleen and increased the percentage of T-cells in mice [49]. The spleens of 10- to 11-month-old male C3B10RF~ mice that received 50% of DR for 2 to 3 months were found to be lighter when compared to the controls. The cell yields in the spleen of those DR mice were lower than the controls {(21 4. 4) × 106 VS. (88 4- 10) × 10 6 per spleenl [49]. The female C3B10RFI mice on a 50% DR also resulted in decreased nucleated cell yields in the spleen [50]. Also, our data showed a 30% and a 34% loss of the S

159 ceils in the young and old DR mice, respectively, as compared to their matched controis. The white blood cell (WBC) count was reported to be decreased by DR [47,51,52]. Total leukocyte counts (cells/mm 3 of blood) in DR mice were reduced by 45% compared to the controls [51]. The total WBC counts of our 30-month-old male B6C3FI mice were 8.17 ± 0.53 and 2.10 + 0.11 for the AI and DR groups respectively (Table II). This indicates that DR mice have only 25% of the WBC counts of the controls. In a similar study, Weindruch and Walford reported that nucleated WBC counts in the DR mice were 17% of the control counts [47]. Taking into account all the above information regarding the effects of DR on immune tissues and our data on proliferative index (reduced percentage of S-cells) in thymus and spleen and the decreased WBC counts in our DR mice, we conclude that the immune tissues of DR mice do not proliferate as much as AL mice, perhaps preserving function. However, through the adaptive stimulation mechanism DR mice might produce more active and functional immune cell types, thus the DR animal's immune system might be functionally younger than the AL cohorts. Thereby, the DR animals may maintain better health and prolong their life. In the case of rats, incidences of leukemia were reported not to be affected by DR [14]. The pathology data collected at NCTR indicates that the incidences of lymphoma in kidney in male B6C3Fl mice at 30 months were 3/15 and 0/15 for AL and DR groups, respectively. A similar lymphoma incidence pattern was present at 36 months when 6/15 AL mice vs. 0/15 DR mice were affected. Thus, age-associated lymphoma incidence appears to be affected by DR [14]. The distribution of S-phase cells among thymus, spleen, and bone marrow contrast the increase in S-cell populations in kidney tissue in old mice in both AL and DR groups. This could have been due to lymphoma, nephropathy, and glomerulosclerosis. These renal lesions might result in tubular damages and in turn may have caused the stimulation of compensatory renal tubular cell proliferation [53-55]. The reduction in the percentage of S-cells in old mice in thymus and bone marrow indicated that DNA synthesis decreased in the organs as aging progressed. The present study showed that in young animals, the thymus tissues and tissues of higher proliferating property such as bone marrow, may be more susceptible to change by DR than in old mice. Also, the data indicates that DR does affect cell proliferation (reduced S-cell population) and its effect depends on (a) the organ (b) age, (c) the duration of DR, and (d) the rate of cell proliferation. ACKNOWLEDGEMENT

The authors wish to thank Mmes. Patricia Rutherford, Denise Armstrong, and Virginia Taylor for their help in editing and typing during the preparation of the manuscript.

160 REFERENCES 1 C.M. McCay, M.F. Crowell, and L.A. Maynard, The effect of retarded growth upon the length of life span and upon the ultimate body size. J. Nutr., 10 (1935) 63-79. 2 N.H. Sakar, G. Fernandes, N.T. Telag, I.A. Koorides and R.A. Good, Low calorie diet prevents the development of mammary tumors in C3H mice and reduces circulating prolactin levels, murine mammary tumor virus expression, and proliferation of mammary alveolar cells. Proc. NatL Acad. Sci. U.S.A., 79 (1982) 7758-7762. 3 E.J. Masoro, Nutrition and aging-a current assessment. J. Nutr., 115 (1985) 842-848. 4 M. Pollard and P.H. Luckert, Tumorigenic effects of direct- and indirect-acting chemical carcinogens in rats on a restricted diet. J. Natl. Cancer Inst., 74 (1985) 1347-1349. 5 A.M. Holehan and B.J. Merry, The experimental manipulation of aging by diet. Biol. Rev., 61 (1986) 329-368. 6 M.W. Pariza and R.K. Boutwell, Historical perspective: Calories and energy expenditure in carcinogenesis. Am. J. Clin. Nutr., 45 (1987) 151-156. 7 R. Weindruch and R.L. Walford, The Retardation o f Aging and Diseases by Dietary Restriction. Charles C. Thomas Publisher, Springfield, IL, 1988. 8 A. Turturro and R.W. Hart, Longevity-assurance mechanisms and caloric restriction. Ann. N.Y. Acad. Sci., 621 (1991) 363-372. 9 M.H. Ross and G. Bras, Lasting influence of early caloric restriction on prevalence of neoplasms in the rat. J. Natl. Cancer Inst., 47 (1971) 1095-1113. 10 A. Tannenbaum, Effects of varying caloric intake upon tumor incidence and tumor growth. Ann. N.Y. Acad. Sci., 49 (1947) 5-18. 11 M.J. Tucker, The effect of long-term food restriction on tumors in rodents. Int. J. Cancer, 23 (1979) 803-807. 12 R. Weindruch, R.L. Walford, S. Fligiel and D. Guthrie, The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. Z Nutr., 116 (1986) 641-654. 13 T.D. Heller, P.R. Holt and A. Richardson, Food restriction retards age-related histological changes in rat small intestine. Gastroenterology, 98 (1990) 387-391. 14 W.M. Witt, W. G. Sheldon and J.D. Thurman, Pathological endpoints in dietary restricted rodentsFischer 344 rats and B6C3F I, mice. In L. Fishbein (ed.), Biological Effects of Dietary Restriction, Springer-Verlag, Berlin, 1991, pp. 73-86. 15 B.J. Merry and A.M. Holehan, In vivo DNA synthesis in the dietary restricted long-lived rat. Exp. Gerontol., 20 (1985) 15-28. 16 H.L. Lipscomb and J.G. Sharp, Effects of reduced food intake on morphometry and cell production in the small intestine of the rat. Virchows Arch. (Cell PathoL), 41 (1982) 285-293. 17 E. Lok, F.W. Scott, R. Mongeau, E.A. Nera, S. Malcolm and D.B. Clayson, Calorie restriction and cellular proliferation in various tissues of the female Swiss Webster mouse. Cancer Lett., 51 (1990) 67-73. 18 D.B. Clayson, F.W. Scott, R. Mongeau, E.A. Nera and E. Lok, Dietary and caloric restriction: Its effect on cellular proliferation in selected mouse tissues. In L. Fishbein (ed.), Biological Effects of Dietary Restriction, Springer-Verlag, Berlin, 1991, pp. 55-64. 19 M.R. Melamed, P.F. Mullaney and M.L. Mendelsohn (eds.), Flow Cytometry and Sorting. John Wiley and Sons, Inc., New York, 1979. 20 J.W. Gray, F. Dolbeare, M.G. Pallavicini, W. Beisker and F. Waldman, Cell cycle analysis using flow cytometry. Int. J. Radiat. Biol., 49 (1986) 237-255. 21 K.R. Geisinger, T.E. Kute, R.B. Marshall, H.D. Homesley and T.M. Morgan, Analysis of the relationships of the ploidy and cell cycle kinetics to differentiation and the female sex steroid hormone receptors in adenocarcinoma of the endometrium. Am..L Clin. Pathol., 85 (1986) 536-541. 22 G. Raschella, A. Negroni, C. Giubilei, A. Romeo, S. Ferrari, M.A. Castello and C. Dominici, Transcription of N-myc and proliferation related genes is linked to human neuroblastoma. Cancer Lett., 56 (1991) 45-51. 23 M.H. Lu, J. Anson, W.G. Hinson, A. Turturro and R.W. Hart, Cell cycle analysis in calorically restricted rats. Proc. Am. Assoc. Cancer Res., 30 (1990) 180.

161 24 25 26 27

28 29 30 31 32 33

34 35 36 37 38 39 40

41 42 43

44 45

46 47 48 49

M.H. Lu, W.G. Hinson, A. Turturro, J. Anson and R.W. Hart, Cell cycle analysis in bone marrow and kidney tissues of dietary restricted rats. Mech. Ageing. Dev., 59 (1991) 111-121. L.L. Vindelov, I.J. Christensen and N.I. Nissen, A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry, 3 (1983) 323-327. P.N. Dean, A simplified method of DNA distribution analysis. Cell Tissue Kinetics, 13 (1980) 299-308. S.L. Cohen, A.W. Rademaker, H.R. Salwen, W. A. Franklin, F. Gonzales-Crussi, S.T. Rosen and K.D. Bauer, Analysis of DNA ploidy and proliferative activity in relation to histology and N-mycamplification in neuroblastoma. Am. J. Pathol., 136 (1990) 1043-1052. G.L. Coleman, S.W. Barthold, G.W. Osbaldiston, S.J. Foster and A.M. Jones, Pathological changes during aging in barrier-reared Fischer 344 male rats. J. Gerontol., 32 (1977) 258-278. L.G. Lajtha, On the concept of the cell cycle. J. Cell. Comp. PhysioL, 62 (1963) 143-145. M. Winick and A. Noble, Cellular response in rats during malnutrition at various ages. J. Nutr., 89 (1966) 300-306. N. Weraarchakui, R. Strong, W.G. Wood and A. Richardson, The effect of aging and dietary restriction on DNA repair. Exp. Cell Res., 181 (1989) 197-204. J.M. Lipman, A. Turturro and R.W. Hart, The influence of dietary restriction on DNA repair in rodents: A preliminary study. Mech. Ageing Dev., 48 (1989) 135-143. V.M. Craddock and J.V. Frei, Induction of liver cell adenomata in the rat by a single treatment with N-methyl-N-nitrosourea given at various times after partial hepatectomy. Br. J. Cancer, 30 (1974) 502-511. S. Preston-Martin, M.C. Pike, R.K. Ross, F.A. Jones and B.E. Henderson, Increased cell division as a cause of human cancer. Cancer Res., 50 (1990 7415-7421. A.W. Pound and L.J. McGnire, Influence of repeated liver regeneration on hepatic carcinogenesis by diethylnitrosamine in mice. Br. J. Cancer, 37 (1978) 595-602. R.M. Hicks, Pathological and biochemical aspects of tumor promotion. Carcinogenesis, 4 (1983) 1208-1214. A.W. Pound, Carcinogenesis and cell proliferation. N.Z. Med. J., 67 (1968) 88-89. S.M. Cohen and L.B. Ellwein, Cell proliferation in carcinogenesis. Science, 249 (1990) 1007-1011. H.L. Gentler and H. Bernstein, DNA change as the primary cause of aging. Q. Rev. BioL, 56 (1981) 279-303. B.N. Ames, R.L. Saul, E. Schwiers, R. Adelman and R. Cathcard, Oxidative DNA damage as related to cancer and aging: Assay of thymine glycol, thymidine glycol, and hydroxymethylaracil in human and rat urine. In R.S. Sohal, L.S. Birbaum and G. Cutler (eds.), Molecular Biology of Aging: Gene Stability and Gene Expression, Raven Press, New York, 1985, pp. 137-144. H.R. Warner and A.R. Price, Involvement of DNA repair in cancer and aging. J. GerontoL, 44 (1989) 44-54. C. Franceschi, Cell proliferation, cell death and aging. Aging, 1 (1989) 3-15. H.M. Rabes, R2 Kerler, C. Schuster, G. Rode, M. Legner, L. Muller and T. Bucher. Cell proliferation and DNA repair in the liver during early stages of chemical carcinogenesis. Adv. Enzyme Regul.. 25 (1985) 241-262. G. Charnley and S. Tannenbaum, Flow cytometric analysis of the effect of sodium chloride on gastric cancer risk in the rat. Cancer Res., 45 (1985) 5606-5616. R.L. Walford, P.J. Meredith and K.E. Cheney, Immune engineering: prospect for correction of agerelated immunodeficiency states. In T. Makinodan and E. Yunis (eds.), Immunology and Aging, Plenum Press, New York, 1977, pp. 183-202. N. Fabris, Immunology and aging. Experientia, 37 (1981) 1041-1043. R. Weindruch and R.L. Walford, The Retardation of Aging and Disease by Dietary Restriction, Charles C. Thomas Publisher, Springfield, IL 1988, pp. 179-197. A. Franklin, S.M. Hinsull and D. Bellamy, The effect of dietary restriction on thymus autonomy. Thymus, 5 (1983) 345-354. R. Weindruch, S.R.S. Gottesman and R.L. Walford, Modification of age-related immune decline in mice dietarily restricted from or after midadulthood. Proc. NatL Acad. Sci. U.S.A., 79 (1982) 898 -902.

162 50

51 52 53 54

55

R.H. Weindruch, J.A. Kristie, F. Naeim, B.G. Mullen and R.L. Walford, Influence of weaninginitiated dietary restriction on response to T-cell mitogens and on splenic T-cell levels in a long-lived mouse hybrid. Exp. Gerontol., 17 (1982) 49-64. R.K. Boutwell, M.K. Bruch and H.P. Rush, Some physiological effects associated with chronic caloric restriction. Am. J. Physiol., 154 (1948) 517-524. C.M. McCay, Chemical aspects of ageing and the effect of diet upon ageing. In A.I. Lansing (ed.), Cowdry's Problem of Ageing, Williams and Wilkins, Baltimore 1952, pp. 139-202. G.E. Striker, R.B. Nagle, P.W. Kohnen and E.A. Smuckler, Response to unilateral nephroectomy in old rats. Arch. Pathol. 37 (1969) 429-442. L.M. Lowenstein, The rat as a model for aging in the kidney. In D.C. Gibson, R.C. Adelman and C. Finch (eds.), Development of the Rodents as a Model System of Aging, DHEW Publication No. (NIH) 79-161, National Institute of Health, 1978, pp. 235-242. N.L.R. Bucher and R.A. Malt, Regeneration of Liver and Kidney, Little, Brown and Company, Boston, 1971, pp. 231-244.