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Knockout Mice
Department of Pathology (K.H.B., J.E.A., M.M.M.), Department of Molecular and Human Genetics (K.H.B., M.M.M.), and Department of Molecular and Cellular Biology (M.M.M.), Baylor College of Medicine, Houston, Texas 77030; and Department of Cancer Biology (P.S.), Dana-Farber Cancer Institute, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Stuart A. Wallace Chair and Professor, Department of Pathology, One Baylor Plaza, Baylor College of Medicine, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu.
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ABSTRACT |
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:ßA and :ßB) endocrine, paracrine, and autocrine factors of the TGFß superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin (Inh) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inh knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1 
S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inh mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inh knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inh lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inh double knockouts. After gonadectomy, 50% of cyclin D2, Inh double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inh single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inh knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland. |
INTRODUCTION |
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Understanding the functions of cell cycle regulators is relevant to cancer biology, development, and physiological cell proliferation in adult tissues. For example, cell division and differentiation in the ovary are essential for female fertility. During follicle development, granulosa cells proliferate in response to FSH, and they abruptly and terminally differentiate after ovulation in response to LH. Female infertility in knockout models lacking cyclin D2 and the CDK inhibitor protein p27Kip1 demonstrate essential roles for each in cell cycle control during ovarian folliculogenesis and lutenization (reviewed in Ref.3). D-type cyclins and p27 are key mediators of the G1 to S phase transition. Cyclin D2-CDK activity is implicated in promoting S phase commitment and DNA synthesis; this is countered by p27 (reviewed in Ref.4). Cyclin D2 knockout females have a defect in granulosa cell proliferation that results in a block in follicle development before antral follicle formation (5). Conversely, infertility in p27 knockout females is associated with defects in granulosa cell cycle withdrawal and differentiation at luteinization. The enlarged ovaries in p27 knockout females are filled with multilayered follicles that do not progress to form corpora lutea (6, 7, 8). Thus, although cyclin D2 and p27 are widely expressed, functional redundancy in most tissues limits the deleterious effects of their loss, and knockout phenotypes indicate essential functions of these regulators in ovarian physiology. In males, cyclin D2 and p27 are not essential for terminal differentiation and function of Sertoli cells or fertility, but both are important regulators of testis growth. Cyclin D2 knockout males have hypoplastic testes with weights 2.5-fold less than wild type (5), whereas p27 knockout males exhibit nearly a 2-fold increase in testicular weight (7).
Inhibins are heterodimeric (:ßA and :ßB) endocrine, paracrine, and autocrine members of the TGFß superfamily that are produced in several types of cells, including those of the adrenal cortex, ovarian granulosa cells, and testicular Sertoli cells (9). They are essential tumor suppressors in mice (10). Inhibin (Inh) knockout mice demonstrate defects in the control of granulosa and Sertoli cell proliferation, and both females and males succumb to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage (10, 11). Originally, these were classified as granulosa/Sertoli cell tumors based on observations of gonadal histology, including 1) Before tumor formation in young Inh knockout mice, precocious proliferation or overt hyperplasia of these cell types can be seen (10); and 2) Well developed tumors in terminal animals develop pathoneumonic features of granulosa/Sertoli cell mixed-sex cord stromal tissues, including insular, microfollicular, and tubule-like patterning (10). In Inh knockout males, decreased numbers of Leydig cells are observed in nontumorous interstitial regions with tumor progression, and germ cells are lost as tumors develop (9, 10). More recently, Affymetrix GeneChip expression profiling analyses have indicated that ovarian tumors from Inh knockout females express a number of granulosa cell markers, including aromatase, estrogen receptor ß, and cyclin D2, but do not express thecal layer markers such as LH receptor and estrogen receptor (Burns, K. H., G. E. Owens, J. H. Nilson, and M. M. Matzuk, unpublished data). The tumor cells also function, to some extent, like granulosa/Sertoli cells in that they require gonadotropin hormones to sustain proliferation (11) and produce steroid hormones and activin dimers (ßA:ßA, ßB:ßB, and ßA:ßB) (9, 10). Tumor activin production promotes a cachexia-like wasting syndrome in the Inh knockout mice; this is associated with elevated levels of circulating FSH, hepatocellular necrosis, and a block in the differentiation of epithelial lineages in the stomach (12, 13, 14). Castrate (gonadectomized) Inh knockout mice develop sex steroidogenic tumors of the adrenal cortex later in life and a similar wasting syndrome (12).
To better understand the roles of cell cycle proteins in growth, function, and tumorigenesis in the gonads and adrenal glands, we are studying how cell cycle regulators may function as genetic modifiers of tumor development in Inh knockout mice. Because of their important functions in regulating cell cycle progression in the ovary, we focused these studies on cyclin D2 and p27. We previously reported that tumorigenesis in the absence of inhibins is accompanied by an increased expression of cyclin D2 mRNA and a decreased expression of p27 protein (15). Moreover, we found that inhibins and p27 cooperate to suppress gonadal tumor development and granulosa cell proliferation, as double-mutant mice lacking both Inh and p27 develop tumors with earlier onset and a more aggressive course than mice lacking Inh alone (15). In the present study, we investigate the roles of cyclin D2 in gonadal tumorigenesis and the potential of both p27 and cyclin D2 to act as modifiers of adrenal tumor development in the Inh knockout mice. This approach leads us to further insights regarding how extracellular factors such as inhibins and gonadotropins normally interface with the cell cycle machinery to regulate cell proliferation and differentiation in these tissues.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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knockout mice and is critical for the physiological proliferation of granulosa cells in response to FSH (3, 5, 15). To assess its role in ovarian tumor development in Inh knockout mice, we generated double-mutant mice lacking both cyclin D2 and Inh. We found that cyclin D2-/-, Inh-/- double-knockout females lived longer than control mice lacking inhibins alone; the majority of double-knockout mice lived longer than 17 wk, as opposed to Inh-/- single-knockout females with 50% survival at between 12 and 13 wk of age. Also, whereas 95% of Inh null females die by 17 wk of age, 95% of double-knockout females had died only after 29 wk. Interestingly, we also noticed an intermediate survival of females that were null at the Inh locus and heterozygous at the cyclin D2 locus (50% survival at between 14 and 15 wk of age; 95% dead at 22 wk) (Fig. 1A
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-/- and cyclin D2-/-, Inh-/- littermates were killed at 6 and 9 wk of age. Histological analyses of tissues from 6-wk-old mice demonstrated advanced disruption of ovarian architecture in cyclin D2+/-, Inh-/- females (Fig. 1B
); serial sections through one of these tumor samples revealed no remaining follicles or oocytes (data not shown). However, in double-knockout mice at 6 wk of age, ovarian histology to the preantral follicle stage remains relatively intact with only minor perturbations observed (Fig. 1C). Notably, in cyclin D2 -/-, Inh-/- females at this age, the absence of inhibins causes follicles to develop more layers of granulosa cells than the four layers that can be observed in single knockouts lacking cyclin D2 (5). Asymmetric granulosa cell proliferation resulting in acentric oocytes was also noted in a subset of these follicles (Fig. 1C). By 9 wk of age, recognizable ovarian tissue was largely replaced with relatively undifferentiated tumor cells, and histological differences between these two genotypes became less clear (Fig. 1, D and E). Grossly, however, ovaries from the double-knockout females were uniformly smaller than those of cyclin D2+/-, Inh-/- at 9 wk (Fig. 1F).
Ovarian Phenotypes in Adult Cyclin D2-/-, Inh-/- and Cyclin D2-/- Females
Although ovarian tumors developed with later onset and less aggressive progression in double-mutant cyclin D2-/-, Inh-/- females as compared with Inh-/- mice, tumors were observed in all 17 of the double-knockout females that were followed. Ninety-five percent of cyclin D2-/-, Inh-/- females included in this study succumbed to tumor formation or were killed in the final stages of their wasting disease by 29 wk of age; the oldest cyclin D2-/-, Inh-/- female died of the wasting syndrome and ovarian tumor development at 39 wk of age (Fig. 1A).
Grossly, tumors recovered from double-knockout females were large, with clear fluid-filled and blood-filled cysts (Fig. 2A). Histologically, 11 of 12 tumors from cyclin D2-/-, Inh-/- females demonstrated features of granulosa cell tumors (Fig. 2B) and also had large expanses of cells that appear relatively undifferentiated. In only one of 12 tumor samples from double-knockout females, Sertoli-like tubule structures were a predominant feature (data not shown). Thus, these tumors are essentially indistinguishable histologically from the ovarian tumors that develop in Inh single-knockout females (10).
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alone (24.9 ± 7.4 ng/ml), vs. 7.9 ± 2.5 ng/ml for 2- to 3-month-old adult wild-type mice (Fig. 2C). Single-knockout females lacking cyclin D2 were killed at 2–3 months of age as controls for these analyses. We found that serum levels of FSH were also measurably elevated in these mice, averaging 16.8 ± 3.8 ng/ml. The absence of cyclin D2 in female mice was associated with a degenerative ovarian phenotype that has not been previously reported (5). Cyclin D2 null females had hemorrhagic and cystic ovaries with dying follicles apparent by 2 months of age (Fig. 2, D and E). By 8 months of age, these cysts had largely recessed, and small ovaries that were devoid of oocytes and ovarian follicles remained (Fig. 2F). However, despite these degenerative changes in the granulosa cell compartments of ovarian follicles in the absence of cyclin D2, the cyclin D2-/-, Inh-/- null females still develop granulosa cell tumors with 100% penetrance.
Cyclin D2 Plays a Critical Role in Testicular Tumor Development and Progression
To study the necessary functions of cyclin D2 in testicular tumor development in Inh-/- mice, we monitored double-mutant males for up to 1 yr. We found that cyclin D2+/-, Inh-/- males rapidly developed fatal testicular tumors as did Inh-/- males, with 50% survival at 9 and 8.5 wk, respectively. In contrast, double-knockout males were protected from early signs of tumor development, and all 17 of the double-knockout male mice survived beyond 26 wk of age. The 50% survival of these cyclin D2-/-, Inh-/- males was between 40 and 41 wk; five of the 17 (29%) were still alive at 1 yr of age (Fig. 3A).
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-/- and cyclin D2-/-, Inh-/- male littermates were killed at 9 wk of age. In cyclin D2+/-, Inh-/- males, those mice surviving to 9 wk already had advanced tumors; testes were bilaterally enlarged and grossly hemorrhagic (Fig. 3B, left). In contrast, cyclin D2-/-, Inh-/- males had hypoplastic testes with no grossly visible tumor foci (Fig. 3B, right). Upon histological analyses, cyclin D2+/-, Inh-/- 9-wk testes showed aberrant testicular morphology, with tubules and interstitial spaces being filled or replaced with proliferating Sertoli cells and blood (Fig. 3C). No regions were without tumor development, and there was no evidence of germ cell production. In cyclin D2-/-, Inh-/- mice at 9 wk of age, there is essentially no disruption of the seminiferous epithelium, and all stages of spermatogenesis were seen in testicular tubules (Fig. 3D).
We also recorded testicular weights in mice of different genotypes at 6 wk of age to investigate the roles of cyclin D2 in testis growth and verify that cyclin D2 is contributing to hyperplasia and early tumor development in the absence of inhibins (Fig. 3E). Even at this relatively early time point, the growth-suppressive effects of loss of cyclin D2 were clear; both cyclin D2 single knockout testes as well as double knockout testes lacking cyclin D2 and inhibins were more than 50% smaller than those of wild-type control mice. Additionally, male mice lacking one cyclin D2 allele had a 20% reduction in testis weight. Histological evaluation of 6 wk-old cyclin D2 -/- testes indicated the presence of all testicular cell types (i.e. germ cells developed through the spermatozoa stage, Sertoli cells, and Leydig/interstitial cells) (Fig. 3, F and G), and we ascribe their reduced size to proportional hypoplasia. This further suggests that cyclin D2 is a determining factor for the regulation of CDK activity and ultimately cell proliferation in the developing testis (see Discussion).
Male Inh knockout mice develop infertility secondary to testicular tumor development, and males with slower tumor progression can retain fertility up to 6–7 wk of age (9). To ascertain the reproductive potential of cyclin D2-/-, Inh-/- males, five double-knockout mice were mated with wild-type females. All of the double-knockout males sired offspring, consistent with the histological presence of spermatozoa in the testes of these mice. Paternal age at the time of birth of the last litter ranged between 4 and 7 months. During this period, double-knockout males sired litters with slightly lower than normal frequency (0.76 ± 0.07 litters/month), but normal litter size (7.4 ± 0.7 pups per litter) compared with control males (
1 litter per month and 8 pups per litter) (16). Thus, delayed tumor development in cyclin D2-/-, Inh-/- males prolongs their reproductive lifespan. These findings are also consistent with the observations that neither cyclin D2 nor Inh deficiency alone functions as a primary cause of infertility in males (5, 9).
Cyclin D2-/-, Inh-/- Male Mice Develop Bilateral Gonadal Tumors
Although cyclin D2 is a potent modifier of testicular tumorigenesis in Inh knockout mice, 12 of the 17 cyclin D2-/-, Inh-/- double-knockout males eventually succumbed to tumor development within 1 yr (Fig. 3A). These double-knockout males exhibited the typical weight loss and cachexia observed in the single Inh knockout mice (Table 1; see below). Upon gross morphological analyses, these tumors involved nearly the entirety of both testes in all cases. Testes were bilaterally enlarged and distorted in shape, with multiple sites of hemorrhage (Fig. 4A). Histological analyses of tumors from seven double-knockout males with overt signs of cachexia revealed typical features of mixed-sex cord stromal tumors (Fig. 4, B–D). Although relatively normal seminiferous tubules were seen displaced to the edge of the testis in some samples, the majority of testicular tissue from these mice was comprised of poorly differentiated and mitotically active tumor cells (Fig. 4B). In some regions of these samples, collections of cells formed nests and annular tubules, histological features described in granulosa cell tumors (Fig. 4, C and D).
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-/- males without overt signs of cachexia at 1 yr of age were killed for an evaluation of tumor progression. Although advanced and hemorrhagic testicular tumors were found in several of these mice, small testes from one double knockout demonstrated minimal tumor development, with a single, relatively restricted solid tumor focus noted unilaterally (Fig. 4E). Thus, although testicular tumor development in Inh-/- mice is seriously compromised in the absence of cyclin D2, inhibins remain essential for long-term tumor suppression.
Characterization of the Expression of Cell Cycle Regulators in Gonadal Tumors
To identify compensations in tumors of cyclin D2-/-, Inh-/- mice that allow for tumor progression in the absence of cyclin D2, we compared the expression of related cyclin and CDK genes within these tumors to gene expression levels in Inh single-knockout tumors and control tissues. We selected cyclin D1, cyclin D3, cyclin E1, cyclin E2, Cdk2, and Cdk4 for Northern blot analyses, given the evidence for their roles in governing the commitment to S phase, and the propensity for cell cycle regulation by modulation of their mRNA (15).
We found that cyclin D3 and cyclin E2 mRNA were not highly expressed in testicular tumors in both Inh-/- and cyclin D2-/-, Inh-/- mice, with values more than 10-fold less and 4- to 5-fold less than wild type for the respective mRNAs (Fig. 5, A and B). Notably, cyclin D3 mRNA levels were enriched in cyclin D2 knockout testes relative to wild-type controls. Changes in Cdk2 expression were not associated with genotype (Fig. 5C). Cdk4 mRNA was up-regulated 4- to 5-fold in tumor samples of Inh-/- mice, as we have previously reported, and this change was comparable to that seen in double-knockout tumor samples (15) (Fig. 5D). Expression of cyclin D1 and cyclin E1 were at the limits of detection in both testes and testicular tumors. Thus, in each case, there was no significant difference in gene expression detected between tumors from mice of Inh-/- and cyclin D2-/-, Inh-/- genotypes. In females, decreased expression of cyclin D3 with ovarian tumor development was also noted, whereas other signals were either too weak to detect (cyclin D1, cyclin E1, cyclin E2) or showed negligible change across the tissue types analyzed (Cdk2 and Cdk4)(data not shown). Although relative increases in cyclin or Cdk mRNAs were not observed in tumors of cyclin D2-/-, Inh-/- mice as compared with those of Inh-/- mice, these findings do not exclude regulatory modifications of cyclin/Cdk protein complexes in the double knockouts that may allow for tumor progression without cyclin D2.
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-/- Mice Develop a Cachexia-Like Wasting Syndrome knockout mice die of a cachexia-like wasting syndrome that results from effects of tumor-produced activins within the glandular stomach and liver (12, 13, 14). To assess the natural course of this syndrome, mice were weighed weekly, and their total body mass recorded. All Inh null mice succumbing to tumor development displayed significant weight loss (average loss of 19–26.2% maximum body weight; Table 1), and most showed marked thoracic kyphoscoliosis and minimal responsiveness when being moved between the scale and their cage. Weight loss paralleled tumor development irrespective of sex or genotype at the cyclin D2 locus (Table 1) and was forestalled in cyclin D2-/-, Inh-/- males as compared with their cyclin D2+/-, Inh-/- littermates and Inh-/- mice (Fig. 6A, and data not shown).
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-/- double-knockout mice recapitulate findings previously reported for Inh single knockouts and provide evidence of the cause of the wasting syndrome (12). There is marked depletion of the parietal cell population within the fundic glandular stomach (Fig. 6, B and C) and hepatocellular necrosis and scattered lymphocytic infiltration in the liver (Fig. 6, D and E).
Castrate p27-/-, Inh-/- Mice Succumb to Adrenal Tumor Development
Early death caused by granulosa/Sertoli cell tumors can be circumvented in mice lacking inhibin by gonadectomy to remove the ovaries or testes. In this experiment, castrate Inh knockout mice can live up to nearly 40 wk (females) or 70 wk (males) before they succumb to adrenal cortical tumors. We hypothesized that cell cycle regulators important in the development of gonadal tumors (i.e. p27 and cyclin D2) may also play roles in adrenal tumor development in the absence of inhibins. Substantiating that there may be similar means of pathogenesis, adrenal tumors in Inh knockout mice are similar to the gonadal tumors by several histological and endocrinological criteria (9, 12). Additionally, we found that similar to gonadal tumors, adrenal tumors in this model demonstrate an up-regulation of Cdk4 mRNA (Fig. 7A) and express appreciable levels of both p27 and cyclin D2 mRNA (data not shown).
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single-knockout mice (Fig. 7B), and survival curves for gonadectomized Inh-/-; p27+/-, Inh-/-; and p27-/-, Inh-/- mice are superimposable in both sexes (Fig. 7, C and D). Mice developed the concomitant wasting syndrome in association with adrenal tumor progression as described previously (12) (data not shown). Mice with overt signs of weight loss and kyphoscoliosis were killed and adrenal tumor tissue was recovered. In the majority of cases, gross evidence of adrenal tumor development was noted only unilaterally (83%), and there was no correlation between cases of bilateral tumors and genotype or sex.
It was found that serum FSH was elevated to a greater extent in gonadectomized p27-/-, Inh-/- double-knockout mice at the time of death as compared with either p27-/- or Inh-/- single-knockout controls (Fig. 7E). In both sexes, double-knockout FSH levels were at or outside the range of the assay (>150 ng/ml) as compared with 48.5 ± 17.2 ng/ml for females and 27.6 ± 7.4 ng/ml for males lacking inhibins alone. Similarly, LH levels were elevated 5-fold in double-knockout females and 11-fold in double-knockout males over average levels recorded for Inh-/- mice in the final stages of adrenal tumor development, although these were not significantly higher than levels measured in gonadectomized wild-type mice or p27-/- single knockouts (Fig. 7F).
Gonadectomized Cyclin D2-/-, Inh-/- Mice Exhibit Prolonged Lifespan
Similar to the experiments described in the previous section, cyclin D2-/-, Inh-/- double-knockout mice were gonadectomized to investigate the functions of cyclin D2 in adrenal tumor development. Mice developing adrenal tumors exhibited signs of the associated cachexia-like syndrome, regardless of genotype, and were killed as the cachexia became severe. Interestingly, although adrenal tumors developed in double-knockout mice of both sexes, cyclin D2-/-, Inh-/- double knockouts exhibited longer life spans than Inh knockout littermates with functional cyclin D2-encoding alleles. After gonadectomy, 50% of cyclin D2, Inh double-knockout males live longer than 46 wk, 10 wk longer than 50% of littermates lacking only inhibins. The rightward shift in the survival curve as compared with Inh single knockouts is significant (P = 0.001) (Fig. 8A).
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-/- mice was more striking in the case of females, which normally develop adrenal tumors earlier than males in the absence of inhibins. Like male double knockouts, 50% of female cyclin D2, Inh double knockouts live to 47 wk before succumbing to adrenal tumor development, and this contrasts sharply with the 50% survival of Inh single-knockout females at between 27 and 28 wk (Fig. 8B). A significant extension of survival was also observed in cyclin D2+/-, Inh-/- females, with 50% survival of these mice at 34 wk of age (P = 0.013). Notably, at 34 wk of age, there was 100% survival of cyclin D2-/-, Inh-/- females and only 14% survival of inhibin knockout females wild type at the cyclin D2 locus. Despite these findings, which indicate important roles for cyclin D2 in adrenal tumor development, tumorigenesis pathways continue without it in both sexes.
The histopathology of adrenal tumors in the cyclin D2-/-, Inh-/- double-knockout mice is not distinguishable from adrenal tumors described previously in Inh knockouts (Fig. 8C). There were no differences in serum gonadotropin hormone levels in cyclin D2-/-, Inh-/- double-knockout mice as compared with Inh single knockouts at the final stages of adrenal tumor progression (data not shown). Thus, inhibins remain essential tumor suppressors in the adrenal cortex, even in the absence of cyclin D2.
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DISCUSSION |
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knockout mice. Toward this end, we have bred the Inh knockout mutant mice with lines of mice harboring targeted mutations in p27 and cyclin D2 to assess the roles of these G1 to S phase mediators in gonadal and adrenal tumor development and progression. We previously reported that p27 cooperates with inhibins to suppress hyperplasia and tumorigenesis in the gonads (15). This is based on the finding that loss of p27 leads to earlier onset of tumors and a more aggressive course of disease in double-knockout mice as compared with controls developing tumors in the absence of inhibins alone.
In the present study, we used a similar strategy and generated double-knockout mice lacking cyclin D2 and Inh. We found that the loss of cyclin D2 delayed the onset and progression of gonadal tumorigenesis and prolonged the life of double-knockout mice as compared with littermate controls lacking inhibins and heterozygous at the cyclin D2 locus.
In the ovaries of female double-knockout mice, this delay in tumorigenesis was also associated with a prolonged presence of oocytes as compared with cyclin D2+/-, Inh-/- females. We hypothesize that oocyte death in the absence of inhibins reflects aberrant signaling from the granulosa cells undergoing transformation, as oocytes are known to rely on communication with these surrounding somatic cells (17). Although we interpret the delayed oocyte loss in cyclin D2-/-, Inh-/- females as an indicator of forestalled tumorigenesis, it should be noted that we cannot rule out effects of the loss of cyclin D2 alone. Interestingly, although this rescue was readily appreciable in females, cyclin D2 proved to be a more striking modifier of testicular tumorigenesis. The reasons for this sexual dimorphism remain unclear, although we speculate that there may be different thresholds for loss of cell cycle control in granulosa and Sertoli cell populations (Fig. 9). We base this not only on the results of the present study, but the observation that we have found testicular tumorigenesis is more readily rescued or forestalled than ovarian tumor development in double-mutant mice lacking inhibins and either 1) lacking FSHß (18), follistatin (19), or the two estrogen receptors (19A ); or 2) ectopically overexpressing inhibin A (:ßA) (20). The converse also holds in that Inh-/- ovarian tumorigenesis is exacerbated by loss of p27 to a greater extent than testicular tumor development (15). The model presented is also consistent with the physiological function of these two types of cells. Under normal conditions, female fertility relies on the controlled proliferation of the granulosa cells within developing follicles, whereas male fertility depends on the establishment of a terminally differentiated Sertoli cell population. In addition to potential differences in cellular proliferation thresholds, qualitatively different mechanisms of tumor development may be involved in the ovaries and testes, representing unique downstream events of inhibin signaling in each tissue. The molecular signaling mechanisms of inhibins and the ultimate intracellular targets of these pathways remain obscure so this is a point of speculation. It seems clear, however, that different means of oncogenesis result in tumors with varying requirements for D-type cyclins, even in the same tissue type (21).
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-/- double-knockout males phenocopies FSHß-/-, Inh-/- mice (18). Similarly, the testicular hypoplasia with preserved gametogenesis in cyclin D2 single-knockout males is not unlike the phenotype of FSHß-/- males (5, 22). Together, these data suggest that cyclin D2 may be a target of FSH not only in granulosa cells as previously reported (5), but also in Sertoli cells. To what extent absence of cyclin D2 or FSHß protects Inh-/- males from tumor development because of effects during testicular development remains a point of speculation. Our findings indicate that not only is the histological appearance of tumor foci clearly delayed by many weeks in the cyclin D2, Inh double-knockout males, but that the progression of adult tumor pathogenesis is also likely slowed. We base this on the observation that the onset of infertility in cyclin D2, Inh double knockouts precedes death by more than 16 wk, a time period longer than the total lifespan of Inh knockout mice. Slowed tumor progression, as opposed to simply delayed onset, strongly suggests a direct role for cyclin D2 in facilitating at least some stages of adult tumor progression. Future studies to compare tumor aggressiveness in mice of different genotypes or to modulate these gene products in a temporal manner may provide additional insights. Thus, the present studies reemphasize the important roles of cyclin D2 as a regulator of physiological testicular growth and call attention to its potential as a key cell cycle mediator in testicular tumor development and gonadotropin hormone responsiveness in such tumors.
Incidental observations of cyclin D2 knockout females in this study revealed a previously unreported degenerative ovarian phenotype. We propose that the block in follicle maturation in cyclin D2-/- females causes aberrant and ineffective gonadal feedback to the central endocrine axis, perhaps a result of deficient steroid hormone or inhibin production (24). Such a disturbance would account for the elevated FSH levels that would secondarily contribute to cystic changes within the ovaries of older mice. Thus, the phenotype may prove similar in many respects to that of the aromatase knockout or estrogen receptor knockout females (25, 26, 27). Additionally, the absence of cyclin D2 may directly cause pituitary abnormalities leading to hypergonadotropism. These are intriguing findings that will hopefully lead to more focused long-term studies of reproductive phenotypes in this knockout. Additionally, we speculate that early elevations in gonadotropin hormones in the absence of cyclin D2 may contribute to sustaining tumor development in the double-knockout females, albeit delayed as compared with Inh-/- mice.
In trying to identify means in which tumors circumvent proliferation pathways requiring cyclin D2, we searched for augmented expression of related cyclins and associated CDKs in tumors of double-knockout mice. We found no differences in the mRNA expression of D- and E-type cyclins, Cdk2 or Cdk4, in these tumors as compared with tumor specimens of Inh-/- mice alone. Up-regulation of Cdk4 in Inh-/- and cyclin D2-/-, Inh-/- testicular tumors, as well as in the adrenal tumors of Inh-/- mice of both sexes, may represent a causative factor in tumor development. Decreased expression of cyclin D3 and E2 may reflect changes in the cellular composition of tumors as compared with normal tissue. In addition to Sertoli cells, cyclin D3 is expressed in Leydig cells and spermatogonia in the adult testis (28). Consistent with this, human granulosa cell cancers and testicular germ cell tumors also express very low levels of cyclin D1 and cyclin D3 (5). The continued development of mouse lines with multiple mutations and conditional null alleles will allow us to pose more questions regarding the redundancy of D- and E-type cyclins and the in vivo significance of their interactions with specific CDKs.
In addition to analyzing the roles of p27 and cyclin D2 in gonadal tumor development, we gonadectomized double-mutant mice to better appreciate their functions in tumors of the adrenal cortex. We found that p27-/-, Inh-/- double-knockout mice succumb to adrenal tumor development in a manner indistinguishable from Inh-/- mice considering animal survival and morphological features of the tumors. We did note, however, that these tumors developed in a measurably different endocrine milieu, so that FSH and LH levels in both sexes are markedly increased in castrate double knockouts with advanced adrenal tumors as compared with single knockouts lacking inhibins alone. In the case of FSH, this effect is unique to double-knockout mice so that castration of p27-/- single knockouts does not reproduce the increase, whereas LH is elevated to nearly comparable levels in gonadectomized mice lacking p27. Although reasons for this dichotomy and the molecular basis for p27/inhibin interactions remain unclear, these studies underscore the potential roles of p27 in modulating gonadotropin production independent of gonadal effects. Tissue transplantation experiments and tissue-specific p27 mutant mice may facilitate future studies to delineate roles of this cell cycle regulator in the pituitary and gonads. Assuming that higher levels of gonadotropins are established in a time frame to influence tumor development and are not reflective of a differential terminal stress response, it is unclear why lack of p27 does not exacerbate the tumor phenotype. Similarly, it is not clear whether p27 normally functions cell autonomously as a tumor suppressor in the adrenal cortex. If p27 has suppressive effects on cell division here, its loss is compensated for in both the p27-/- single-knockout and p27-/-, Inh-/- double-knockout mice by other factors that are more effectual in the adrenal gland than in the gonads. Candidates that may have such an activity include p57Kip2 and p21Cip1; their loss has already been associated with adrenal cortical tumors (29, 30).
In contrast to p27, which does not obviously influence adrenal tumor progression in the Inh-/- context, loss of cyclin D2 prolongs the lifespan of castrate cyclin D2, Inh double knockouts. After gonadectomy, 50% survival of male and female cyclin D2-/-, Inh-/- mice is extended 10 wk and 20 wk, respectively, compared with Inh-/- single-knockout controls. Thus, cyclin D2 is likely to be acting as a proliferative factor within the adrenal cortex contributing to tumorigenesis. Although no phenotype has been previously described in cyclin D2 heterozygotes, our data indicate that cyclin D2+/-, Inh-/- females have a significantly prolonged lifespan before dying of ovarian or adrenal tumor development as compared with Inh-/- females. Moreover, we found that cyclin D2+/- males have a measurable reduction in testicular weights at 6 wk of age as compared with wild-type males. These findings call attention to gene dosage effects and consequences of cyclin D2 haploinsufficiency. In contrast, testicular germ cell tumors in humans are not only associated with increased cyclin D2 mRNA levels, but also increased copy number of the 12p13 region, which includes the cyclin D2 locus (5). Thus, these studies further emphasize dosage effects of cyclin D2 alleles.
In conclusion, our studies have uncovered important, tissue-specific, and sexually dimorphic functions of cell cycle regulators in tumorigenesis. The first knockout studies of these factors attuned researchers to their necessity in specific tissues and physiological processes; our work assigns them recognized roles in cancer pathophysiology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(Inha), cyclin D2 (Ccnd2), and p27Kip1 (Cdkn1b) mutant mice have been described previously (5, 7, 10). Mice were genotyped by Southern blot analyses of genomic tail DNA. Inh genotyping protocols are described (10). Modified protocols were used for cyclin D2 and p27 genotyping, using EcoRV and EcoRI enzyme digests, respectively, and the Southern blot probes listed in Table 2. This results in a 11.8-kb band for the wild-type cyclin D2 allele and a 8.4-kb band for the mutant cyclin D2 allele. For p27 genotyping, this results in a 7.5-kb band for the wild-type allele and a 4.0-kb band for the targeted allele. Double-knockout mice were identified in litters from matings of double heterozygous mutant females (cyclin D2+/-, Inh+/-; p27+/-, Inh+/-) and double-mutant males (cyclin D2-/-, Inh+/-; p27-/-, Inh+/-). Both cyclin D2-/-, Inh+ and p27 -/-, Inh -/- double-knockout mice were obtained at approximately the expected 1:8 Mendelian ratio with similar numbers of male and female double knockouts being produced. All lines of mice were maintained on a mixed C57BL/6J/129S6/SvEv genetic background.
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Weight Data and Survival Curves
Mice were followed weekly for tumor development by measuring total body weight and monitoring for kyphosis as described (12). Mice were killed when they developed overt signs of cachexia. Statistical significance was determined by Student’s t test comparing weeks of age at death.
Morphological and Histological Analysis
Mice of each sex and genotype were killed and tissues were collected immediately into 10% buffered formalin (ovaries) or Bouin’s fixative (testes). After overnight fixation, tissues were embedded in paraffin blocks, sectioned, and stained with hematoxylin and eosin (if not otherwise indicated) or periodic acid Schiff (PAS) and hematoxylin. Embedding and staining were performed by standard procedures in the Baylor College of Medicine Pathology Core Services laboratory.
Serum Analyses
Mice were anesthetized by isoflurane inhalation (Abbott Laboratories, North Chicago, IL), and blood was recovered by closed cardiac puncture. Serum was separated by centrifugation in Microtainer tubes (Becton Dickinson, Franklin Lakes, NJ) and stored at -80 C before analysis. FSH and LH RIAs were performed in the University of Virginia Ligand Core Facility. Methods can be found on their website at http://www.healthsystem.virginia.edu. For this manuscript, data are provided as ± SEM and statistical significance was assessed by ANOVA.
RNA isolation and Probe Preparation
For RT-PCR and Northern blot analysis, tissues from killed animals were immediately preserved in RNAlater reagent (Ambion, Inc., Austin, TX) before RNA extraction. Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction using the RNA STAT-60 reagent according to the instructions of the manufacturer (Leedo Medical Laboratories, Houston, TX). RT-PCR was used only for probe preparation and was not employed as a means to quantify mRNA expression. cDNA was prepared from wild-type ovary RNA by RT-PCR using the Superscript system (Invitrogen, Carlsbad, CA). Sequences of selected cell cycle regulators were then amplified according to the manufacturer’s instructions using primers designed based on the sequences available in the National Center for Biotechnology Information public database (Table 2). The PCR products were ligated into T vector (Promega Corp., Madison, WI), checked by bidirectional sequencing, and used as templates for the synthesis of Northern blot probes. In addition, colleagues provided several cDNA probes as generous gifts.
Northern Blot Analysis
For Northern blot analyses, 15 µg of each RNA sample were electrophoresed and transferred to nylon membrane. Tumor tissue and RNA samples were not pooled, and each lane represents RNA isolated from a single mouse. Probes radiolabeled with [32P]dATP were synthesized using the Strip-EZ kit (Ambion, Inc.). Autoradiography and PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA) allowed for visualization and quantification of probe hybridization, respectively. After washing, membranes were exposed to X-OMAT film (Eastman Kodak Co., Rochester, NY) with the use of intensifying screens overnight. PhosphorImager plates were scanned and analyzed using ImageQuant software (Molecular Dynamics, Inc.). A background level for each membrane was subtracted, and blots were then stripped and reprobed for Gapd to allow for loading corrections.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CDK, Cyclin-dependent kinase; Inh, inhibin ; PAS, periodic acid Schiff.
Received for publication February 5, 2003. Accepted for publication July 3, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-Inhibin is a tumor-suppressor gene with gonadal specificity in mice. Nature 360:313–319[CrossRef][Medline]
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