Minimal effects of dietary restriction on neuroendocrine carcinogenesis in Rb+/– mice

Z.Dave Sharp1,5, Wen-Hwa Lee1, Alexander Yu Nikitin4, Andrea Flesken-Nikitin4, Yuji Ikeno2, Robert Reddick3, Arlan G. Richardson2 and James F. Nelson2

1 Departments of Molecular Medicine,
2 Physiology, and
3 Pathology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX, and
4 Department of Biomedical Sciences, Cornell University, Campus Road T2 007 VRT, Ithaca, NY 14853-6401, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The efficacy of dietary restriction in retarding tumor growth is well established in rodents. However, gene and cell lineage specificity of dietary restriction effects is far less defined. Mice with a single copy of the retinoblastoma susceptibility gene (Rb) develop a well-established syndrome of mouse neuroendocrine neoplasia associated with Rb deficiency. Thus, if DR represses tumor growth in this model, it should be unambiguously attributed to the Rb defect in neuroendocrine cell lineages. To address this possibility, Rb+/– mice were entered into a diet restriction study. Surprisingly, 40–50% reductions in dietary intake, relative to an ad libitum group, started on either postnatal day 28 or 42 had little to no effect on either the frequency or growth of pituitary tumors either during the latency period (postnatal day 224) or at the time of their natural death. Consistent with cross-section data, survival of 65 diet restricted Rb+/– mice was almost identical to that of 67 Rb+/– mice fed ad libitum (AL); median life span was 414 and 436 days for AL and DR groups, respectively. These findings indicate that diet restriction provides no significant benefit in delaying growth and progression of neuroendocrine tumors exhibiting loss of RB function. They also introduce the possibility that RB is required for the tumor-repressive effects of DR.

Abbreviations: DR, diet restriction; EAP, early atypical proliferation.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In rodents, diet restriction (DR) is one of the best and most effective procedures for repressing tumor growth (1,2). In radiation-induced cancers, restricted diet intake significantly reduced the incidence of solid tumors, and/or lymphatic (3,4) or myeloid (5) leukemia. A large body of evidence has also demonstrated a similar reduction in chemically induced tumors (reviewed in ref. 6). A study of spontaneous tumors in rodents showed that DR reduced the relative incidence and delayed the onset of pituitary tumors, adrenal pheochromocytomas, pancreatic islet cell tumors and interstitial cell tumors of the testis (7). Interestingly, food restriction initiated at 6 months of age in Fischer 344 rats was as effective as restriction begun at six weeks of age in delaying neoplasia (8). Overall, the preponderance of evidence indicates that DR provides a protective effect in tumor development and growth.

These studies provided evidence that DR plays a critical role in delaying tumor formation in animal models either spontaneously initiated or chemically induced. Recent studies on genetically modified mice are in good agreement with the earlier observations. In a v-Ha-ras transgenic (Onco) mouse model, food restriction was beneficial in extending survival. Mammary tumor incidence was reduced, and alterations in expression of cytokines, oncogenes (including p53) and free radical scavenging enzymes were all consistent with a protective effect of DR (9). Recently, DR was shown to delay tumor formation in mice nullizygous for p53 (10,11). Interestingly, the age-specific cancer death rate of ad libitum (AL):DR ratios were 4.3 for p53–/– and 4.4 in p53+/+ mice (11). This observation is complicated by the fact that lymphomas are common tumors in mice regardless of genotype. Thus, despite the accelerated onset of carcinogenesis in p53–/– mice, the tumor-delaying effect of DR was similar in the two genotypes (11). These data indicated that DR, in this model, operates independent of p53 making it difficult to dissect the DR effect on tumor formation predisposed by genetic factors.

Mice heterozygous for deletion of the retinoblastoma susceptibility gene (Rb+/–) may be useful in addressing gene and cell lineage specificity of DR benefits in reducing tumor incidence for a number of reasons. Briefly, Rb+/– mice die at about 12 months of age due to melanotroph tumors of the pituitary gland (1216). Consistent with a syndrome of multiple neuroendocrine neoplasia, it was demonstrated that early Rb-deficient cells are detectable in the intermediate and anterior lobe of the pituitary, the thyroid, parathyroid glands, and the adrenal medulla by three months of postnatal development of Rb+/– mice (17). In the pituitary, the early foci formed by early atypical proliferation (EAP) are observable with complete penetrance after the postnatal cessation of melanotroph proliferation at about postnatal day (P) 30 to P60. By 6 months, all mice produce melanotrophic tumors that begin to invade their surrounding tissues. At ~12 months of age they begin to die from compression of brain structures by pituitary tumors or the trachea by thyroid tumors. Thus, high penetrance, synchronicity, and neuroendocrine character of carcinogenesis associated with Rb deficiency, as well as low frequency of those tumors in unmodified laboratory mice, make Rb+/– mice an attractive model for evaluating the significance of gene and cell-lineage-specific context for DR efficacy in reduction of tumor incidence. Interestingly, in this model, in contrast to other models tested, DR has a markedly reduced ability to override the strong genetic defect that predisposes tumor development and growth in Rb+/– mice.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and dietary protocols
Male Rb+/– mice (12) were used in this study. This strain is estimated to be 75% C57BL and 25% 129. The mice housed four to a filter-topped microisolator cage were under the care and supervision of the technical staff of the Animal Core of the Nathan Shock Center of Excellence in Basic Biology of Aging. Animals were kept on a cycle of 12:12 h dark:light (lights on at 05:00 h). The procedures and experiments involving use of mice were approved by the Institutional Animal Care and Use Committee and are consistent with the NIH Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Education, the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act (National Academy Press, Washington, DC).

For the first 4–6 weeks, all mice were fed ad libitum (AL) Harlan Teklad LM-485 mouse/rat sterilizable diet 7912 (Madison, WI). At 6 weeks, a randomly selected group assigned to the cross-sectional studies were allowed to continue on the AL diet until sacrifice at postnatal day (P) 224. Another group of mice was fed 60% of the mean food intake of the AL Group until sacrifice at the same time. Food intake by AL mice was measured once a week and the amount ingested per day calculated. Intake was corrected for food not eaten (i.e. found in the bottom of the cage, 30.75%). DR mice were given their food allotment 1 h before the start of dark phase of the light cycle. The mice entered into the survival studies were treated exactly the same, except that they were restricted to 50% of the mean food intake of Group AL beginning at 4 weeks postnatally.

There were some differences that were noted between Rb+/– and wild type mice. First, Rb+/– mice are slightly larger than wild-type littermates, as previously reported (18). Although the difference is not large (10%), it is reproducible. Second, since 40% restriction of the DR group of Rb+/– mice demonstrated only a 20% weight reduction, we therefore considered the possibility that this level of diet restriction in this pedigree was not sufficient to observe beneficial effects on tumor growth in Rb+/– mice. In a small group of mice, it was determined that this strain well tolerated a 50% reduction in calories. It was also determined in the same test group that diet restriction started earlier at 4 weeks of age was well tolerated. Accordingly, 50% reduction in food starting at P28 was used in the survival studies.

Genotyping and pituitary histology
The identification of Rb+/– mice by PCR has been described previously (19). In the cross-sectional studies, pituitary glands from Rb+/+ and Rb+/– mice were fixed by immersion in Bouin’s fluid, dissected under a stereo operation microscope (Nikon) and prepared for serial sectioning and sequential three-dimensional reconstruction of the specimens (19).

Pathological assessment
In the survival studies, a complete pathological analysis of the mice after their natural death included the following: gross and microscopic analysis of all visible tumors as well as brain, pituitary gland, heart, aorta, lung, trachea, esophagus, stomach, small intestine, colon, liver, gall bladder, pancreas, spleen, urinary bladder, thyroid/parathyroid gland, psoas muscle, sternum, spinal cord, vertebrae, knee joint, nasal passage, thymus, ventral abdominal skin and gonadal tissue including the testes, preputial gland, epididymis, prostate and seminal vesicle. Any other organs or tissues with gross lesions were also examined. The heart, lung, kidneys, testes, liver, spleen, brain, pancreas, adrenal glands were weighed and the dimensions and mass of visible tumors were described. After the gross inspection, all of the tissues were fixed immediately in 10% neutral buffered formalin. The fixed tissues, which are processed conventionally, were embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin–eosin. A profile of pathological lesions was constructed for each mouse that included the prevalence of both neoplastic and non-neoplastic diseases. The size of each tumor, tumor burden (the number of different tumors in a mouse), overall incidence of each disease and incidence of fatal disease (probable causes of death) were measured.

Statistical analyses
Logrank test of the survival curves and unpaired t-tests were performed using GraphPad Prism (Ver. 3.0, Graphpad Software).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To determine the effects of DR on tumor growth, Rb+/– mice were randomly assigned to a group (n = 11) that was fed 60% of the food consumed by the AL group (n = 15). DR was imposed beginning on P42 in both groups. Four Rb+/+ mice fed ad libitum were also included in this initial study. At about 8 months, an age when tumor development was previously observed to be well advanced, mice in all groups were killed, the pituitaries dissected, fixed and prepared for gross and microscopic examination. This analysis was done without prior knowledge of the genotype and experimental group. An analysis of the pituitary was chosen for this cross-sectional study since tumor initiation and development are well understood, and are representative of the cancers in other neuroendocrine organs. The results of the microscopic examination of the cross-sectional specimens are summarized in Table IGo. All pituitaries in the 11 DR and 15 AL mice demonstrated tumors at varying stages of development. As expected, the wild type mice fed ad libitum were all EAP and tumor free. Topographic analysis of the pituitaries showed that the 15 AL Rb+/– mice had a total of 29 intermediate lobe tumors compared with the 21 observed in 11 DR mice. Of these, it was noted that the AL group had nine EAPs or small tumors that spanned only one level of the pituitary compared with one in the DR group. However, this was offset by those that spanned two levels or more in the DR group. None of these differences were statistically significant by t-tests. In the anterior lobe, there were a total of four tumors in four mice in the AL group and eight tumors in six mice in the DR group. Combined the AL group had 33 tumors compared with 29 in the DR group, differences that are not statistically significant.


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Table I. Histological evaluation of pituitary tumors in RB+/- mice on P224
 
Although 40% DR did not appear to have an effect on overall pituitary tumor development and growth to P224, there could be delayed growth as the animals age, and there could also be beneficial effects on other neuroendocrine tumor growth. To address this possibility, Rb+/– mice were randomly assigned to an AL group (n = 67) and a 50% diet-restricted group (n = 65) beginning at P28 for a survival study. Figure 1Go shows the comparison of weight data for 50% DR and AL groups entered into the survival studies. These data indicate that a reduction of one half the ad libitum food intake results in a 30% reduction in weight in the Rb+/– strains. This is in line with the 32% decrease in weight observed in male B10C3F1 mice receiving 56% of the ad libitum intake begun at 12 months of age (20). The survival curve in Figure 2Go shows that, consistent with the cross-sectional data indicating no difference in growth of pituitary tumors at P224, there is no statistical difference in survival of the two groups. The median life spans for the AL and DR are 414 and 436 days, respectively. The maximum life spans for mice in the AL and DR groups were 616 and 574 days, respectively.



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Fig. 1. Response of Rb mice to a 50% reduction in food intake. DR (+/–) and AL (+/–), n = 16; AL (+/+), n = 4. The diet reduction protocol is described in Materials and methods.

 


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Fig. 2. Survival of ad libitum and 50% diet restricted Rb+/– mice. Median life span was 414 and 436 for AL and DR groups, respectively. P = 0.0768 for the logrank comparisons of the survival curves.

 
Autopsies were performed to assess the development and progression of both of neuroendocrine tumors and C-cell tumor metastases (17), and to obtain an overall gross examination of the mice at death. The results (Table IIGo), consistent with the cross-sectional analysis, show that there are no differences (unpaired t-tests) in any of the values presented in Table IIGo. Consistent with Nikitin et al. (17), lung metastasis were evident in the histological sections in both groups with no statistical differences (unpaired t-tests) in the frequency between the DR and AL groups. The lower percentage of mice with lung metastases (39% compared with 84%) (17) is probably due to the variance in detection methods. In an earlier study (17) calcitonin immunostaining was used to detect very small tumors. Our current study was based on evaluation of hematoxylin and eosin slides only. The probable causes of death were pituitary and/or thyroid tumors in both groups with no statistical difference in the frequencies. The cause of death of one mouse in the DR group was undetermined. There was a tendency for the DR tumors to be smaller in size, although this difference did not translate into longer survival for this group. Overall, the necropsy data are consistent with survival data, both of which show that DR has little if any affect on tumor formation in the Rb+/– model.


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Table II. Pathology of Rb+/- survival mice at necropsy
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In most previous studies examining the effects of diet restriction on cancer in rodents, beneficial effects have been reported. Of particular relevance to our study were those done with the p53–/– model of murine cancer (10). Although lymphomas developed earlier in p53–/– animals and diet restriction extended their survival, the same margin of improvement was observed in p53+/+ mice, leading the authors to conclude that the mechanism underlying the beneficial effects of diet restriction were independent of their genotype (11). The authors also argued that DR does not require an intact p53 function. Interestingly, in the Rb+/– model, if there is a benefit to diet restriction, it is dramatically reduced compared with others. An advantage of the Rb+/– model over the p53–/– model is the temporal and spatial predictability of tumor development in the Rb model allowed for detailed topographic analyses of the pituitary tumors. While there was no overall difference in the total number of pituitary tumors, it is curious that there were more intermediate lobe tumors in the AL group and more anterior lobe tumors in the DR group. While the differences are not large, we wonder if there is a cell-specific and/or hormonal response to the diet restriction. In previous studies, excessive production of {alpha}-MSH in Rb+/– mice, consistent with intermediate lobe tumors, was documented (21). With the exception of {alpha}-MSH, over production of no other functionally active hormones by the pituitary tumors are known (13,17). Interestingly, {alpha}-MSH is an anorexic hormone (22), and should, if anything, augment the imposed dietary restriction of Rb+/– mice. It is important to note that it was the Rb–/– tumors that did not respond to the energy restriction. This is in contrast to the other somatic tissues (presumably Rb+/–), which did respond as evidence by less weight gain. In addition, anterior lobe tumors are present in less 50% of the mice. Thus, it seems unlikely that a generalized hormonal change counteracted the diet restriction specifically for the tumors.

In this and previous studies, it was determined that Rb+/– mice have a larger mass than wild type. Interestingly, mice that over express Rb are smaller than wild type (18,23). In each case, the change in size is proportional, with no excess accumulation of fat in the larger Rb+/– mice. Thus, it is likely that diet restriction or Rb+/– mice achieves an energy balance equivalent to other models since we calculated the diet restriction based on the ad libitum consumption of the control Rb+/– mice. However, it is still possible that increasing the level of restriction above 50% (as used in the survival study) might have a greater beneficial effect on Rb+/– mice.

The survival curve and median life span are slightly improved for the DR group, but are not statistically significant. Thus, if DR is having a positive effect in the Rb+/– model, it is dramatically attenuated compared with other models such as the p53–/– mice. It does not seem likely that this difference is due to variations in genetic background. Both Rb+/– and p53–/– models have predominantly C57BL background with less than a 25% contribution from the 129 strain. It is interesting that estrogen-induced pituitary prolactin-producing tumors in the ACI rat, unlike Fisher 344, is one other example, albeit strain- and tissue-specific, of carcinogenesis that is also not inhibited by dietary restriction (24,25).

Early atypically proliferating groups of cells are first detectable from 30 to 90 days after birth (17,19). In the pituitary, intermediate lobe cells cease proliferation at about P60. It is, therefore, likely that LOH in these cells occurred prior to P60. DR was imposed in the cross-sectional group at P42 and at P28 in the survival studies. Thus, it is likely that LOH had occurred and EAPs had begun to form at or around the time that DR was started. In previous detailed studies, the majority of the animals died of brain compression due to large pituitary tumors. However, they can also die from tracheal compression by thyroid C-cell cancers that also develop in this model (17). Thyroid cancers are first detected at about P35-60 as parafollicular proliferates that are comparable with the pituitary EAPs that appear about the same time (17). However, Nikitin et al. (17) reported that the latency period and extent of progression of the thyroid tumors is different from those in pituitary tumors at the time of death. For example, only thyroid tumors demonstrate metastases to the lungs (84% at P380) that are first noticeable as calcitonin-positive cells at about P250 (17). DR and retardation of tumor growth is well established (21,26,27). Of relevance to the present study is retarded growth and accelerated apoptosis of human prostate cancer cells transplanted in diet-restricted rats, effects associated with decreased levels of endocrine IGF-1 (28). Retarded growth rates of bladder cancer in diet-restricted mice are abrogated upon treatment with IGF-1. Thus, it was expected that DR of Rb+/– mice would at least slow the growth of proliferates and subsequent tumors, and delay the progression to the metastatic state. However, it is apparent that chronic 50% DR imposed at as early as P28 had little, if any, effect on either. This finding raises a fundamental question: why are Rb-deficient tumors unresponsive to a 50% reduction in available energy sources?

Despite years of research, little is understood at the molecular level concerning which of the many steps in carcinogenesis is influenced by reduced dietary intake. Speculation has centered on decreases in oxidative stress (29) and reductions in DNA synthesis, and an increase in apoptosis (30,31) as contributing factors. It has also been proposed that the cancer preventive effects of reduced dietary intake are related to lower circulating levels of insulin like growth factor 1, IGF-1 (6,21). The data here imply that Rb may be required for tumor repressive effects of DR. If so, what could be the mechanism? Rb has at least three major roles in cells. First is its role in cell cycle regulation, both the G1 to S transition through E2F interactions (32) and mitosis through its interactions with proteins such as mitosin (33) and HEC (34). Second, it has a positive role in cell differentiation through its interactions with key developmental transcription factors (35). Third, it has been reported to be repressive for transcription catalyzed by RNA polymerases III and I (36). When Rb is over expressed in mice, a dwarf phenotype with elevated circulating IGF-1 is observed (23), suggesting that Rb is somehow involved in IGF-1 responsiveness in growth. Perhaps its absence in Rb-deficient tumors renders them refractory to beneficial effects of lower IGF-1 levels? Responses to IGF-1 mitogenic signaling are balanced with available nutrient levels by mTOR, mammalian target of rapamycin (3741). Rapamycin inhibits the cell cycle and Rb phosphorylation (42,43). It is thus possible that the absence of Rb function disrupts the delicate balance controlling cellular responses to growth factors and nutrient levels mediated by mTOR. If so, this could have important implications for new rapamycin-based anticancer drugs such as CCI-779 (44).

Restoration of Rb function in pituitary tumors leads to their regression and a 20% increase in survival (45). In addition, lung metastases from C-cell thyroid tumors were reduced in Rb+/– mice treated intravenously with Rb gene therapy (17). These studies demonstrated the effectiveness of Rb as a tumor suppressor, even at later stages of tumor development and progression. It would, therefore, be interesting to test if DR provides an additional extension of survival in mice receiving Rb treatment.

In summary, this study has identified one important class of tumors whose response to DR is markedly abridged. Understanding why Rb-deficient tumors have a reduced response to reductions in energy sources may help elucidate the signaling pathways that allow other classes of tumors to respond to restricted diets. The reduced response of neuroendocrine-derived tumors in Rb+/– mice to DR is reminiscent of aggressive small cell lung cancer, which is also of neuroendocrine origin and Rb-deficient. Thus, the poor response of the mouse tumors may be a reflection of this class of deadly neuroendocrine cancers.


    Notes
 
5 To whom correspondence should be addressed at: Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas, USA Email: sharp{at}uthscsa.edu. Back


    Acknowledgments
 
The authors would like to express their gratitude to Vivian Diaz and her staff at the Nathan Shock Animal Core for their expert care of the mice. We are also grateful to the anonymous reviewers for their thoughtful suggestions. This work was support in part by grants from the American Institute of Cancer Research (ZDS), the NIH (ZDS, WHL, AYN, AGR and JFN), with additional support from the Nathan Shock Center for Excellence in the Biological Research.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 25, 2002; revised August 9, 2002; accepted September 22, 2002.