Inhibin and p27 Interact to Regulate Gonadal Tumorigenesis

Sherry C. Cipriano, Lei Chen, Kathleen H. Burns, Andrew Koff and Martin M. Matzuk

Departments of Pathology (S.C.C., L.C., K.H.B., M.M.M.) Molecular and Cellular Biology (M.M.M.), and Molecular and Human Genetics (K.H.B., M.M.M.) Baylor College of Medicine Houston, Texas 77030
Program in Molecular Biology (A.K.) Memorial Sloan-Kettering Cancer Center New York, New York 10021


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tumor suppressors function as antiproliferative signaling proteins, and defects in these genes lead to uncontrolled cell proliferation and cancer. For example, absence of the tumor suppressor p27Kip1, a cyclin-dependent kinase inhibitor (CKI), results in increased body size, hyperplasia of several organs including the testes, and cancer in mice. Similarly, lack of inhibins, {alpha} heterodimeric members of the transforming growth factor-ß (TGFß) superfamily, causes testicular and ovarian tumors of the granulosa/Sertoli cell lineage beginning at 4 weeks of age and adrenal tumors in gonadectomized mice. Neither the cell cycle alterations in the absence of inhibin nor the cause of the increased testis size in the p27 knockout mice is known. To study the molecular (cell cycle) changes that result from absence of inhibins, we analyzed the regulation of cell cycle proteins in gonadal tumors derived from inhibin {alpha} knockout mice (Inha-/-). Northern blot analyses demonstrate that cyclin-dependent kinase 4 (Cdk4) and cyclin D2 mRNA levels are elevated, and immunohistochemistry shows that p27 protein levels are decreased in both ovarian and testicular tumors from Inha-/- mice. These findings suggest that increased Cdk4/cyclin D2 (positive) activity and decreased p27 (negative) activity is causal for gonadal tumor formation. To test this hypothesis, we generated double mutant mice lacking both p27 and inhibin {alpha} to determine whether the tumor suppressors p27 and inhibin have additive suppressor activity in the gonads. Like Inha-/- mice, p27-/-Inha-/- mice demonstrate elevated serum activin levels, ovarian and testicular tumors, and a resultant lethal cachexia-like syndrome. However, whereas 95% of the Inha-/- female mice die by 18 weeks of age, 100% of the p27-/-Inha-/- female mice are dead by 8 weeks. Similarly, 95% of the Inha-/- single mutant males die by 13 weeks while 100% of the p27-/-Inha-/- male mice die by 10 weeks. Moreover, tumor foci in p27-/-Inha-/- mice can be observed as early as 2 weeks of age in males and as early as 4 weeks in females. These findings demonstrate that absence of both inhibin and p27 in mice causes earlier development of ovarian and testicular tumors and earlier death compared with absence of inhibin alone.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell proliferation is mediated by a delicate balance between stimulatory and inhibitory signals that function at defined stages of the cell cycle. Among the proteins that regulate each cell cycle stage are the cyclins, cyclin-dependent kinases (Cdks), and cyclin-dependent kinase inhibitors (CKIs). Cell proliferation is dependent upon activation of cyclin D-Cdk4/6 and cyclin E-Cdk2 complexes (1). Deregulation of cell cycle events such as overexpression of cyclins and/or Cdks or underexpression of CKIs can lead to uncontrolled cell proliferation and malignancy. Two distinct families of CKIs exist, the Cip/Kip and Ink4 families (2, 3, 4, 5, 6, 7, 8, 9). The Cip/Kip family consists of p21Cip1 (3), p27Kip1 (9), and p57Kip2 (7). CKIs have been shown to be involved in DNA damage checkpoints and can function as rate-limiting regulators at the G1 to S phase transition by binding to and inactivating cyclin/Cdk complexes. For example, p27Kip1 interacts with cyclin D and cyclin E complexes to inhibit their activity in vitro (10).

Several knockout mouse models lacking cyclins, Cdks, or CKIs have been created to study cell cycle control in vivo. Among these, mice lacking cyclin D2 and p27 exhibit gonadal defects. Female cyclin D2 knockout mice are sterile due to a failure of granulosa cell proliferation, while male mice have decreased testis size (11). p27-deficient mice have larger than normal body size and display intermediate lobe pituitary hyperplasia (12, 13, 14). p27-deficient males have hyperplastic testes (12, 13, 14) and benign prostatic hyperplasia (15), yet are fertile; p27-deficient females are infertile due to defects in cell cycle withdrawal and corpus luteum formation (12, 13, 14).

Our laboratory is interested in the gonadal growth-regulatory properties of the inhibins and activins, which inhibit and stimulate, respectively, pituitary FSH synthesis and secretion in adult mammals (16, 17). We previously generated transgenic mice lacking inhibin {alpha} and therefore functional inhibins ({alpha}/ßA or {alpha}/ßB heterodimers of the TGFß superfamily that share ß- subunits with the activins) (18). Deletion of inhibin {alpha} leads to development of ovarian and testicular granulosa/Sertoli cell tumors in these mice, identifying inhibin as a critical negative regulator of gonadal cell proliferation. Tumor development is accompanied by a cancer cachexia-like wasting syndrome (19) that mimics the cachexia syndromes found in human cancer patients (20, 21). This wasting syndrome was found to be due to oversecretion of activins from the tumors (18, 19). Elevated levels of FSH in inhibin-deficient mice suggested to us that FSH may be a modulator of the tumorigenic process. Indeed, double knockout mice deficient in FSH and inhibin demonstrate a delay in tumor formation and a reduction in the cachexia symptoms compared with mice lacking only inhibin (22).

Extracellular signals control mammalian cell proliferation principally during the G1 phase of the cell cycle. TGFßs, for example, are potent growth inhibitors that suppress cell proliferation and induce cell cycle arrest by multiple mechanisms during the G1 phase (23, 24). p15Ink4b expression and cell cycle arrest are induced this way in human keratinocytes treated in vitro with TGFß1 (5). Activin has been implicated in cell cycle arrest in some cell types. For example, the HepG2 human hepatoma cell line has been shown to be growth arrested by activin A. In addition, activin A further induced accumulation of hypophosphorylated Rb in HepG2 cells preventing progression to the S phase (25). Another study revealed that in mouse B cell hybridoma cells activin A caused G1 cell cycle arrest (26). Similarly, pituitary adenoma cells in primary culture were growth inhibited after treatment for 24 h with activin, and p21Cip1 was up-regulated after 4 h treatment in a dose-dependent manner (27). However, activin function appears to be different in the gonads where activins can stimulate growth of gonadal tumor cell lines from inhibin-deficient mice (28). Little is known about the inhibin signal transduction cascade, how inhibins are linked to cell cycle regulation, or how inhibins effect growth suppression in the gonads.

In this manuscript, we demonstrate that several cell cycle regulators are altered in the ovaries and testes in the absence of inhibin, and that aberrant expression of these factors is key in loss of growth control and tumor formation. Gonadal tumors that develop in the absence of inhibin show increases in Cdk4 and cyclin D2 mRNA and a decrease in p27 protein expression as compared with normal gonads. Furthermore, absence of the two tumor suppressors, p27 and inhibin, results in earlier tumor development and death, indicating that inhibin and p27 cooperate in regulating gonadal tumor formation and progression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cdk4 and Cyclin D2 Are Increased in Gonadal Tumors
To determine whether there are any cell cycle alterations in the absence of inhibin, we assessed whether up-regulation or down-regulation of a number of cyclins, Cdks, and/or CKIs was associated with the uncontrolled proliferation in the gonads of Inha-/- mice. Northern blot analysis showed only a slight increase in cyclin D2 mRNA between ovarian tumors from Inha-/- mice compared with wild-type ovaries (Fig. 1Go). However, we found a 6-fold increase in cyclin D2 mRNA in testicular tumors of Inha-/- mice compared with wild-type testes (Fig. 1Go). Cdk4 mRNA levels were elevated 3-fold in ovarian and testicular tumors from Inha-/- mice when compared with wild-type ovaries and testes (Fig. 1Go). When mRNA levels of cyclin D2 and Cdk4 in the ovaries and testes of 4-, 8-, and 12-week-old Inha-/- mice were compared, no temporal difference in either cyclin D2 or Cdk4 expression was found, suggesting that inhibin’s effects are early (data not shown). In contrast, levels of p18Ink4c, p19Ink4d, cyclin B1, cyclin B2, and cyclin D3 were increased in wild-type testes alone when compared with wild-type ovaries and gonadal tumors from Inha-/- mice (data not shown). We were not able to detect any significant change in mRNA levels of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, p57Kip2, cyclin A2, cyclin D1, or Cdk2 in gonadal tumors of Inha-/- mice, as compared with normal tissue (data not shown). Thus, inhibin may normally act to inhibit cyclin D2 and Cdk4 and thereby regulate gonadal growth and proliferation (see below).



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Figure 1. Northern Blot Analysis of Cyclin D2 and Cdk4 mRNA in Mouse Testes and Ovaries

Higher expression of cyclin D2 (cyc D2) and Cdk4 mRNA is present in ovarian and testicular tumors from individual 12- to 14-week-old Inha-/- mice when compared with wild-type (WT) ovaries from adult mice. 18S rRNA was used as control for RNA loading.

 
Gonadal Tumors of Inha-/- Mice Have Decreased p27 Expression
The inhibitory effects of TGFßs on p27 expression are mainly at the posttranscriptional level (29). Therefore, we analyzed the levels of p27 protein in the gonads of wild-type and Inha-/- male and female mice by immunohistochemistry. In normal adult mouse and human testes, Sertoli cells (and Leydig cells) stain for p27, which is consistent with the terminally differentiated state of these cells (30). We hypothesized that Sertoli cell tumors present in Inha-/- mice might have reduced p27 protein due to the lack of differentiation and high proliferative activity of these cells. Indeed, by immunohistochemical analysis, p27 was found to be low to undetectable in the Sertoli cell component of Inha-/- ovarian and testicular tumors (Fig. 2Go, B and C, and E and F). Testes from wild-type males at 6 weeks of age showed p27 staining in the Sertoli cells and Leydig cells (Fig. 2DGo; Ref. 30). In contrast, p27 expression in Sertoli cell tumor nodules of Inha-/- male testes had minimal or no p27 protein expression (Fig. 2EGo). Even though p27 expression was absent or barely detectable in Sertoli cell tumors of 6-week-old Inha-/- testes, we observed p27 staining of normal Leydig cells in these same tissues (Fig. 2FGo; Ref. 30). Staining of testes from adult p27-/- mice showed no expression of p27 (data not shown; Ref. 30). The findings in the ovaries of the Inha-/- mice were also consistent. As expected, we observed expression of p27 in the interstitium, primordial follicles, and corpus luteum of 6-week-old wild-type female mice (Fig. 2AGo and data not shown; Ref. 31). We also observed p27 expression in normal appearing primordial follicles and the interstitium of ovaries from Inha-/- mice at 6 weeks of age (Fig. 2BGo). However, in these same ovaries, undifferentiated Sertoli-like tumor cells had very low or undetectable levels of p27 (Fig. 2CGo). Thus, these results suggest a correlation between granulosa cell and Sertoli cell loss of p27, lack of terminal differentiation, and uncontrolled proliferation.



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Figure 2. Immunohistochemical Analysis of p27 Expression

A, Wild-type (WT) ovary showed p27 expression in the interstitium and primordial follicles (expression in the corpus luteum is not depicted). Terminally differentiated Sertoli cells express high levels of p27. While p27 expression was detected in normal appearing primordial follicles and the interstitium of 6-week-old Inha-/- mice (B), undifferentiated Sertoli-like tumor cells in this same ovary were not or barely stained for p27 (C). D, Sertoli cells in the testes of adult wild-type (WT) mice stained for p27. However, Sertoli-like tumor cells in 6-week-old Inha-/- testes minimally or did not stain for p27 (E), while normal-appearing Leydig cells in this same tissue did stain for p27 (F). Magnification is 10–40x.

 
p27-/-Inha-/- Mice Develop Cachexia and Have a Decreased Survival Rate
To determine the in vivo relationship between p27 and inhibin in gonadal tumorigenesis, we generated double mutant mice deficient in both of these genes. An early indicator of ovarian and testicular tumor development in Inha-/- mice is severe weight loss and a cachexia-like wasting syndrome that eventually kills the mice (19). Therefore, to monitor tumor development in p27-/-Inha-/- mice, we weighed wild-type, Inha-/-, p27-/-, and p27-/- Inha-/- mice weekly for 4–20 weeks. As expected, p27-/- male and female mice had higher weight averages beginning at 4–10 weeks of age when compared with wild-type mice (Fig. 3Go, A and B; Ref. 13). Male and female p27-/-Inha-/- mice gained weight initially as did wild-type mice up to 6 weeks of age, but rapidly lost weight thereafter. The cachectic appearance of the p27-/-Inha-/- mice was similar to the Inha-/- mice except that it occurred at an earlier stage and progressed more rapidly (Fig. 3Go, A and B). Serum hematocrit levels of p27-/-Inha-/- males and females revealed that they were anemic, similar to Inha-/- mice (Ref. 18 and data not shown).



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Figure 3. Body Weights of Male (A) and Female (B) Wild-Type, p27-/-, Inha-/-, and p27-/-Inha-/- Mice

Mice were weighed weekly on the same day between 4 and 20 weeks of age. Wild-type, male n = 8, female n = 5; p27-/-, male n = 19, female n = 16; Inha-/-, male n = 20, female n = 20; p27-/-Inha-/-, male n = 17, female n = 3.

 
The p27-/-Inha-/- male and female mice have decreased survival compared with p27-/- or Inha-/- single knockout mice. All of the p27-/-Inha-/- male mice were dead by 10 weeks of age (0% survival), whereas at 10 weeks, 56% of the male Inha-/- mice were still alive (Fig. 4AGo). Similarly, all female p27-/-Inha-/- mice died by 8 weeks of age (0% survival) vs. a 90% survival of the female Inha-/- mice at 8 weeks (Fig. 4BGo). As expected, the survival rate for wild-type and p27-/- male and female mice was 100% during this 4- to 20-week period (Fig. 4Go, A and B, and data not shown). In total, we have generated 21 male and 9 female p27-/-Inha-/- mice. The males have been generated at the expected Mendelian ratio of 1:16. However, the number of viable p27-/-Inha-/- female mice was lower than expected over this same time period, possibly due to early tumor-induced death before genotype analysis.



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Figure 4. Survival Curves of Wild-Type, p27-/-, Inha-/-, and p27-/-Inha-/- Male (A) and Female (B) Mice

Mice were counted once per week between 4 and 20 weeks of age. Wild-type, male n = 8, female n = 5; p27-/-, male n = 5, female n = 14; Inha-/-, male n = 20, female n = 18; p27-/-Inha-/-, male n = 17, female n = 9. Wild-type mice had a 100% survival rate from 4 to 20 weeks.

 
p27-/-Inha-/- Mice Develop Bilateral Hemorrhagic Testicular and Ovarian Tumors
To study the cause of the weight loss and reduced survival of the p27-/-Inha-/- mice, we performed pathological examination of the p27-/-Inha-/- male and female mice at various time points. We found that gonadal tumors developed sooner and were more progressive in p27-/-Inha-/- mice compared with Inha-/- mice. By 7.5 weeks of age, testes of p27-/-Inha-/- mice were grossly enlarged and hemorrhagic compared with wild-type testes (Fig. 5AGo). In contrast to Inha-/- mice, which developed either unilateral or bilateral tumors, all p27-/-Inha-/- male mice analyzed developed bilateral gonadal tumors. Furthermore, these bilateral gonadal tumors in the p27-/-Inha-/- mice were grossly evident by 4 weeks of age in males and 4.5 weeks of age in females.



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Figure 5. Morphological and Histological Analysis of Testes from Male Wild-Type and Mutant Mice

A, Morphological comparison of testes from wild-type (upper part of panel) and 7.5- week-old p27-/-Inha-/-(lower part of panel) mice. Testicular tumors were grossly enlarged and contained areas of hemorrhage (white arrowhead). B, Histological analysis of testes at high-power magnification from a 2-week-old p27-/-Inha-/- mouse demonstrated early Sertoli tumor cell arrangement (starred area) adjacent to normal appearing seminiferous tubules. C, Testis from a 4- week-old p27-/-Inha-/- mouse at low power magnification showed approximately 50% of the tissue is occupied by a single nodular tumor mass consisting of undifferentiated granulosa cells. Some hemorrhage (H) is evident. D, Testis at high-power magnification from a 5.5-week-old p27-/-Inha-/- mouse showed both undifferentiated Sertoli (starred area) and granulosa cell (G) tumor with hemorrhage (H). E, At high power, gland-like structures were seen (black arrows) among the granulosa tumor cells. F, Mitotically active (black arrows) granulosa cell tumor taken from a 8- to 9-week-old p27-/-Inha-/- mouse is shown at high power.

 
Histological examination of the testes of p27-/-Inha-/- mice revealed that tumor formation was first evident in some males at 2 weeks of age as focal lesions (Fig. 5BGo). Strikingly, by 4 weeks of age, approximately 40–50% of each p27-/-Inha-/- testis was composed of single or multiple tumor nodule(s). The masses consisted of approximately 90% granulosa cell tumor cells (Fig. 5CGo). This is in contrast to testicular tumors in 4-week-old Inha-/- mice, which had rare, small nodule lesions of undifferentiated tumor cells (18). While mixed granulosa/Sertoli cell tumors were seen in some p27-/-Inha-/- male mice at 5 weeks of age (Fig. 5DGo), most tumors consisted of primarily granulosa cells (Fig. 5CGo). In one 8-week-old p27-/-Inha-/- male mouse, we observed gland-like structures in the testicular tumor (Fig. 5EGo). This histopathological feature had never been observed in any Inha-/- male tumors. Similar structures are present in the gonads of human patients with Peutz-Jeghers syndrome, which is characterized by oral mucocutaneous hyperpigmentation, intestinal hamartomas, and an increased risk for intestinal and extraintestinal malignancies including gonadal tumors (32). The p27-/-Inha-/- testicular tumors from 8- to 9-week-old males consisted predominantly of mitotically active granulosa tumor cells (Fig. 5FGo) with limited areas of normal tubules located at the periphery of the testis (not shown).

The p27-/-Inha-/- mutant females were also grossly affected at an early age. In contrast to wild-type mice or Inha-/- mice [which have histological evidence of tumors only at an early age (18)], ovaries from p27-/-Inha-/- female mice at 4.5–5.5 weeks of age could show up to a 10-fold increase in the size of the ovary with areas of punctate hemorrhage (Fig. 6AGo and data not shown). Histologically, we found no evidence of normal follicles in the ovaries examined at 5.5 weeks of age (Fig. 6BGo) in contrast to Inha-/- ovaries at the same age (18, 33). Sertoli cell tumors were a prominent component of the tumor phenotype in Inha-/- ovaries. Ovaries from p27-/-Inha-/- mice consisted of only 0–10% Sertoli cell tumor with the majority of the tissue being granulosa cell tumor (Fig. 6CGo). Multiple hemorrhagic cysts (Fig. 6CGo) and significant mitotic activity (Fig. 6DGo) were also seen in the p27-/-Inha-/- ovarian tumors. Thus, absence of p27 increases the rate of development of testicular and ovarian tumors in inhibin {alpha} knockout mice.



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Figure 6. Morphological and Histological Analysis of Ovaries from Female Mice at 5.5 Weeks of Age

A, Ovaries from a p27-/-Inha-/- mouse showed gross enlargement (lower part of panel) compared with wild-type ovaries (upper part of panel). B, Histological analysis at low power of one ovary from panel A showed no normal follicles remaining; instead the ovary consists of primarily granulosa cell tumor with only about 1–10% being Sertoli-like tubule tumor cells. Also, note areas of hemorrhage. C, An ovary from a p27-/-Inha-/- mouse is shown at higher power demonstrating both Sertoli-like tumor (starred area) and granulosa (G) tumor cells along with hemorrhage (H). D, At high power, the undifferentiated granulosa cells are mitotically active as evident in this ovary from a p27-/-Inha-/- mouse.

 
FSH Levels in p27-/-Inha-/- Mice Are Not Significantly Increased When Compared with Inha-/- Mice
Inha-/- mice have increased serum FSH due to loss of local and circulating inhibin (18). Mice lacking both inhibin and FSH had prolonged survival compared with mice lacking only inhibin (22), demonstrating that FSH acts as a modifier of tumor development and the cachexia-like syndrome. To determine whether the earlier death in our p27-/-Inha-/- mice could be due to higher FSH levels or an earlier increase in the FSH levels, we performed FSH enzyme-linked immunosorbent assays (ELISAs). FSH serum levels from p27-/-Inha-/- male mice at an early time point (4 weeks of age) were 195 ± 30.0 ng/ml (n = 3), which was comparable to Inha-/- male mice at the same age (188.1 ± 32.3, n = 5) (Table 1Go). The p27-/-Inha-/- male mice at 7–9 weeks of age (n = 7) had similar FSH levels of 240.7 ± 22.6 ng/ml when compared with Inha-/- 8-week-old male level 257.7 ± 28.1 ng/ml; n = 7. These levels were elevated compared with 4- and 8-week-old male p27-/- mice (75.7 ± 10.98, n = 4 and 78.8 ± 39.3, n = 4, respectively). For unknown reasons, these FSH levels in the p27-/- male mice were higher when compared with wild-type 8-week-old male mice (49.25 ± 3.09 ng/ml, n = 8).


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Table 1. Serum FSH Levels in Wild-Type and Mutant Mice

 
The FSH levels in the 4- to 8-week-old p27-/-Inha-/- female mice (n = 4) were 216 ± 25.5 ng/ml and were elevated compared with 6-week-old wild-type females (22.5 ± 6.07 ng/ml, n = 7) but comparable to Inha-/- females at 6 weeks of age (242.5 ± 24.0 ng/ml, n = 6) (Table 1Go). Similar to males, 6 week-old female p27-/- mice had unexplained increases in FSH levels (61.2 ± 7.33, n = 5) compared with the wild-type control females. Thus, while FSH serum levels are increased in p27-/-Inha-/- male and female mice compared with wild-type mice, they are not significantly different than age-matched serum FSH levels in Inha-/- male and female mice. This suggests that FSH is not directly causal for the earlier death of the p27-/-Inha-/- mice compared with Inha-/- mice.

p27-/-Inha-/- Mice Have Elevated Activin Levels
Gonadal tumors of Inha-/- mice secrete excess activin A and B, which contributes directly to the wasting syndrome (19, 34, 35). To assess this feature of cachexia in p27-/-Inha-/- mice, we determined the serum activin A levels. At an early timepoint, 4-week-old Inha-/- and p27-/-Inha-/- male mice had comparable serum activin A levels that were modestly elevated over controls (Fig. 7AGo). However, p27-/-Inha-/- male mice at 7–9 weeks of age (a timeframe when most of these mice were cachectic and dying) had significantly elevated levels of circulating activin A (55.1 ± 12.6 ng/ml) when compared with 8-week-old wild-type males (0.41 ± 0.10 ng/ml) and 8-week-old p27-/- males (0.12 ± 0.01 ng/ml) and were about 2-fold higher than 8-week-old Inha-/- mice (29.4 ± 8.05 ng/ml) (Fig. 7AGo). Similarly, p27-/-Inha-/- female mice at 4–7 weeks of age (also a timeframe when most were cachectic and dying) had significantly higher activin A levels (18.4 ± 2.1 ng/ml) compared with 6-week-old wild-type (0.28 ± 0.7 ng/ml), 6-week-old p27-/- (0.22 ± 0.06 ng/ml), 4-week-old Inha-/- (4.83 ± 6.0 ng/ml), and 6-week-old Inha-/- (3.79 ± 0.86 ng/ml) females (Fig. 7BGo). As expected, activin A levels of 12- to 14-week-old cachectic Inha-/- male (68.5 ± 11.7 ng/ml) and female Inha-/- mice (84.1 ± 17.4 ng/ml) were also elevated and served as internal controls (Fig. 7Go, A and B; Ref. 18). These findings suggest that the early onset of gonadal tumors in the p27-/-Inha-/- mice results in early increases in activin A levels, earlier and more severe cachexia, and a resultant earlier death of these mice as compared with Inha-/- mice (see below).



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Figure 7. Serum Activin A Levels for Male (A) and Female (B) Mice

Activin A levels for male 8-week-old wild-type (n = 8), 8 week-old p27-/- (n = 8), 4-week-old Inha-/- (n = 7), 8-week-old Inha-/- (n = 11), 12- to 14-week-old Inha-/- (n = 8), 4- to 5-week-old p27-/-Inha-/-(n = 4), and 7- to 9-week-old p27-/-Inha-/-(n = 9) are shown. Female 6-week-old wild-type (n = 7), 6-week-old p27-/- (n = 9), 4-week-old Inha-/- (n = 6), 6-week-old Inha-/- (n = 5), 12- to 14-week-old Inha-/- (n = 8), and 4- to 8-week-old p27-/-Inha-/-(n = 4) are represented.

 
Stomach and Liver Pathology Are Similar in p27-/-Inha-/- and Inha-/- Mice
The high serum activin levels in the Inha-/- mice are a direct cause of severe liver and stomach pathology (19, 34, 35). This pathology includes liver atrophy (i.e. destruction of cells around the central vein and diffuse areas of inflammation) and a block in differentiation of several cell types in the glandular stomach including parietal cells. Both livers and stomachs of male and female p27-/-Inha-/- cachectic mice had equivalent, but not more severe, pathology (data not shown) than cachectic Inha-/- mice (19, 34, 35). Thus, the earlier development of the gonadal tumors in the p27-/-Inha-/- mice and the accompanying elevated activin levels cause a similar but earlier-appearing cachexia-like syndrome compared with mice lacking only inhibin.

Cdk2 Activity Is Increased in p27-/-inha-/- Ovarian Tumors
Levels of p27 in a particular cell type correlate with proliferative activity. The previous Northern blot and immunohistochemical data (Figs. 1Go and 2Go) and the early appearance and rapid progression of ovarian and testicular tumors in p27-/-Inha-/- mice (Figs. 3Go and 4Go) suggested to us that Cdk activity may be increased as well. To test this, we first compared levels of gonadal Cdk4 mRNA in p27-/- vs. p27-/-Inha-/- mice. Cdk4 mRNA expression was slightly higher in the testis of p27-/-Inha-/- males compared with p27-/- male mice (Fig. 8AGo). Due to the low number of mice that were generated, ovaries from p27-/-Inha-/- females were not available for a similar analysis. We next analyzed Cdk kinase activity from the gonads of these mice to determine whether increased Cdk activity was stimulating tumor progression. Unfortunately, attempts to assay Cdk4 and Cdk6 kinase activity in mouse gonadal extracts were not successful (our unpublished data). However, we were able to analyze Cdk2 activity using in vitro kinase assays from individual gonads of male and female wild-type and mutant mice. We found that Cdk2 activity was increased in ovarian tumors from 5.5-week-old p27-/-Inha-/- mice when compared with ovaries from 4-week-old wild-type, Inha-/-, and p27-/- mice (Fig. 8BGo). However, there appeared to be no difference in Cdk2 activity among testes isolated from wild-type, Inha-/-, p27-/-, or p27-/-Inha-/- mice (Fig. 8BGo). Taken together, the Northern blot analysis and in vitro kinase assays suggest that inhibin and p27 predominantly act through increased Cdk2 and Cdk4 activity in the ovaries, and through increased Cdk4 activity in the testes.



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Figure 8. Northern Blot Analysis of Cdk4 Expression and Cdk2 Kinase Activity Assay in Mouse Ovaries and Testes

A, Testicular tumors from 5.5-week-old p27-/-Inha-/- mice had higher expression of Cdk4 mRNA when compared with testes from p27-/- mice. 18S rRNA was used as control for RNA loading. B, Cdk2 activity was higher in ovarian tumor protein extract from 5.5-week-old p27-/-Inha-/- mice as compared with adult ovaries from wild-type (WT), p27-/-, and Inha-/- mice (top). There was no detectable difference in Cdk2 activity among testes from wild-type (WT), -/-, p27-/-, Inha-/-, and p27-/- Inha-/- mice.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cyclins, Cdks, and Cdk inhibitors have been shown to affect cell proliferation and differentiation, and mouse models have greatly contributed to our understanding of the functions of the genes in vivo. Cyclin D2-deficient female mice are infertile due to the lack of granulosa cell proliferation in the ovaries upon stimulation by FSH; cyclin D2-deficient males have hypoplastic testes (11). Cdk inhibitors have been found to be mutated or deleted in a wide range of sporadic tumors, and a decrease in p27 expression has been shown to correlate with poor prognosis in a number of cancers including human colon and breast carcinomas (36, 37), hyperproliferative responses to mitogenic stimulation, and pituitary hyperplasia and adenomas (38). It is possible to study redundancy or cooperativity of gene functions by generating double knockout mice. For example, mice lacking both tumor suppressors p18 and p27 were larger in overall size than p27-deficient mice alone and developed pituitary adenomas at a faster rate (38) linking p18 and p27 in a cooperative cell proliferation control pathway. Using a similar approach, we have generated mice lacking both inhibin and p27 and show that these two tumor suppressors act together to prevent gonadal tumor formation and progression. In this study, p27-/-Inha-/- mice develop tumors faster than mice lacking either inhibin or p27 alone. Also, the earlier development of the tumors results in earlier activin production, earlier symptoms of the activin-induced cachexia syndrome, and earlier death, with all of the p27-/-Inha-/- females dying by 8 weeks and all of the males dying by 10 weeks.

The inhibin signaling pathway is not well characterized, and inhibin receptors have only recently been identified (39, 40). Thus, it was important for us to determine whether inhibin regulates cellular proliferation by mechanisms resembling those of other TGFß family members. We have shown that inhibin acts to negatively regulate p27 protein synthesis similar to the TGFßs. We have also shown that cyclin D2, which is expressed at high levels in some human ovarian granulosa cell tumors (11), is also increased slightly in the Inha-/- ovarian tumors and markedly in the Inha-/- testicular tumors. At this point, it is unclear whether the cyclin D2 elevation is due directly to the absence of inhibin or rather is caused by an elevation in FSH (a positive regulator of cyclin D2 in granulosa cells) (11). Furthermore, we have demonstrated that Cdk4 mRNA is modestly elevated in the Inha-/- ovarian and testicular tumors, and Cdk2 kinase activity is increased in p27-/-Inha-/- ovaries. This has led us to formulate a model wherein inhibins, p27, FSH, cyclin D2, and Cdk2 and/or Cdk4 interact to regulate granulosa cell and Sertoli cell proliferation in vivo (Fig. 9Go).



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Figure 9. A Model for the Role of Inhibin in Cell Cycle Control

Inhibin is known to inhibit the synthesis and secretion of FSH. FSH has been shown to stimulate cyclin D2 synthesis in granulosa cells, and p27Kip1 blocks Cdk activity. We believe that inhibin functions as a tumor suppressor by promoting the formation of inactive cyclin-Cdk-CKI complexes. In the absence of inhibin, we have shown that FSH serum levels increase, cyclin D2 levels increase, Cdk4 mRNA levels increase, and p27 levels decrease in granulosa cells and Sertoli cells. Absence of both p27 and inhibin increases Cdk2 activity in the ovary. Combined absence of p27 and inhibin is expected to increase the formation of active cyclin D2/Cdk4 and cyclin E1/Cdk2 complexes and thereby stimulate granulosa cell and Sertoli cell proliferation. The cooperative action of inhibin and p27Kip1 is necessary to ensure appropriate control of the cell cycle in granulosa cells and Sertoli cells, preclude tumorigenesis or hyperplasia, and allow for terminal differentiation. FSH is a modifier of tumorigenesis in male and female inhibin {alpha} knockouts. It is unclear whether the increase in cyclin D2 levels in inhibin {alpha} knockout mice is a direct effect of absence of inhibin or an indirect effect of increases in FSH.

 
We propose that testicular hyperplasia and infertility in p27-deficient male and female mice, respectively, and tumor formation in both Inha-/- and p27-/-Inha-/- mice result from similar mechanisms, namely lack of cell cycle arrest and incomplete differentiation. Withdrawal from the cell cycle marks differentiation in most cell types, and p27 and inhibin contribute to the induction and maintenance of cell cycle arrest in the granulosa/Sertoli cell lineage. Our study shows that while p27 expression is present in normal differentiated cells of the ovary and testis, protein levels were very low or not detectable in tumor cells of the ovaries and testes in the Inha-/- mice (Fig. 2Go). This suggests that as tumorigenesis progresses in the absence of inhibin, p27 levels decrease whereas p27 continues to be expressed in the adjacent normal-appearing cells.

Interestingly, we found that p27-/-Inha-/- mice develop only bilateral tumors and that tumors in both sexes include a disproportionately large granulosa cell component (as opposed to the Sertoli cell component predominance in Inha-/- tumors) (18). The combined absence of both p27 and inhibin may disrupt important developmental programs that normally direct granulosa/Sertoli cell fate and ensure gonadal genesis. Perhaps steroid hormones produced by these tumors affect this process. Estrogen signaling is important in maintaining granulosa cells, and ovaries of ER{alpha}ß double knockouts show granulosa to Sertoli "transdifferentiation." Markers of Sertoli cell differentiation, including Müllerian inhibiting substance, SGP-2, and Sox9, may be differentially expressed in tumors of Inha-/- mice vs. p27-/-Inha-/- (41). Also, compared with the Inha-/- tumors, ovarian and testicular tumors from p27-/-Inha-/- mice were much more hemorrhagic. Perhaps an increase in angiogenic factors in the absence of both p27 and inhibin also contributes to this hemorrhage and aggressive tumor growth.

Cdk2 activity was found to be increased in ovarian tumors from p27-/-Inha-/- mice as compared with ovaries from wild-type, Inha-/-, and p27-/- mice. However, there was no detectable difference in Cdk2 activity from testes of wild-type, Inha-/-, p27-/-, or p27-/-Inha-/- mice. The significance of this discrepancy between the sexes is not clear. It is possible that this reflects differences in the tissue-specific functions of this particular Cdk (42). It is also conceivable that enhanced gonadal Cdk2 activity in p27-/-Inha-/- females is measurably deleterious to these mice, contributing to their early and acute demise even in comparison to male double knockout counterparts. The increased Cdk2 activity seen in p27-/-Inha-/-ovarian tumors could be due to a decrease in p27 and/or increases in cyclin D2 and Cdk2 protein levels.

Since 100% of the inhibin {alpha} knockout mice develop tumors, yet all Sertoli or granulosa cells do not become tumor cells, undetermined factors most likely affect a cell’s commitment to a tumorigenic path. Overexpression of cyclin D2 and Cdk4, coupled with inactivation of p27, could result in sequestration and functional deficiencies of other Cip/Kip inhibitors. Our studies are important as they define for the first time a link between inhibin and cell cycle proteins in the pathogenesis of ovarian and testicular cancers.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of Mice and Genotyping
Generation of {alpha}-inhibin heterozygous (Inha+/-), inhibin homozygous (Inha-/-) (18), and p27 heterozygous (p27+/-) (13) mice have been described. Inha+/- mice were crossed with p27+/- mice to generate offspring that were homozygous mutant for both p27 and inhibin (p27-/-Inha-/-). p27 is the product of the Cdkn1b gene. The inhibin {alpha} genotype of these offspring was determined using PCR amplification of tail DNA as described (43). The p27 genotype was determined using either Southern blot analysis or PCR. Primers used in the PCR reactions were specific for the targeted p27 allele (primer1: 5'-AGGTGAGAGTGTCTAACGG-3'; primer2: 5'-AGTGCTTCTCCAAGTCCC-3'; primer3: 5'-GCGAGGATCTCGTCGTGAC-3'). All three primers were added to each PCR reaction yielding either wild-type (130 bp), p27 mutant (450 bp), or both bands.

Weight Data and Serum Analysis
Mice were weighed once per week for a period of 4 to 20 weeks. To determine the significance of the survival rates, a confidence interval test was used. For serum analyses, mice were anesthetized using Metofane (Schering Plough Animal Health Corp., Union, NJ), and serum was collected into Microtainer tubes (Becton Dickinson and Co., Franklin Lakes, NJ) using cardiac puncture. Activin A (44) and FSH (43) mouse serum levels were determined using ELISA methods. A one-way ANOVA test was used to determine the statistical significance of these results. Values are reported as mean ± SEM.

Morphological, Histological, and Immunohistochemical Analysis
Tissues were fixed overnight in 10% neutral buffered formalin, embedded, sectioned, and stained with hematoxylin and eosin or periodic acid Schiff as described (44). Morphological and histological analyses were performed on at least 21 male and 5 female p27-/-Inha-/- mice. Immunohistochemical analyses of p27 expression in mouse ovaries (31) and testes (30) were performed as described previously. Tissue sections of 4–5 µm were used for these studies.

Northern Blot Analysis
Total RNA was isolated from testes and ovaries of wild-type, p27-/-, Inha-/-, and p27-/-Inha-/- mice using the TRI-Reagent method (45). Fifteen micrograms of total RNA were run in each lane, and blots were hybridized (45) with their respective probes. Blots were stripped by boiling in a solution of 0.5x SSC and 1.0% SDS for 15 min and then rehybridized with an 18S rRNA control probe. Signals detected on these blots were quantified using the Molecular Dynamics, Inc. photodensitometer (Sunnyvale, CA).

Kinase Assay
Individual mouse ovaries and testes were snap frozen in liquid nitrogen and homogenized on ice with a Dounce homogenizer. Protein lysates were harvested by centrifugation at 4 C at 10,000 rpm for 15 min. Protein concentration was determined as described previously (46). Kinase activity was determined by immunoprecipitation of protein with either polyclonal anti-Cdk2, anti-Cdk4, or anti-Cdk6 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by Histone H1 (Roche Molecular Biochemicals, Indianapolis, IN) phosphorylation assay for Cdk2, or RB (Santa Cruz Biotechnology, Inc.) phosphorylation assay for Cdk4 and Cdk6 (46). Signals detected on blots were quantified using the Molecular Dynamics, Inc. photodensitometer (Sunnyvale, CA).


    ACKNOWLEDGMENTS
 
The authors would like to thank the following investigators for their generous gifts: Dr. Steven Elledge for pBSmcyclinD1, pBSmcyclinD2, pBSmcyclinD3, and pBSmp27; Dr. Charles Sherr for pBSmp15 and pBSmp16; and Dr. Hideyo Yasuda for pmCdk2 and pmCdk4. We also thank Ms. Michele Park for excellent advice and critical discussions regarding this manuscript and Dr. T. Rajendra Kumar for critical review of the manuscript. We thank Drs. Anthony Lau and Hua Chang for assistance with computer graphics and Ms. Shirley Baker for assistance in manuscript preparation.


    FOOTNOTES
 
Address requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Professor and Stuart A. Wallace Chair, Department of Pathology, One Baylor Plaza, Baylor College of Medicine, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu

This work was supported in part by NIH Grant CA-60651 (to M.M.M.) and a National Research Service Award 1F32-CA-81746–01 from the National Cancer Institute (to S.C.C.). K.H.B. is a student in the Medical Scientist Training Program and is supported in part by NIH Grant T32GM-07330 and Grant T32EY07102 from The National Eye Institute.

Received for publication January 3, 2001. Revision received February 23, 2001. Accepted for publication March 8, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Sherr CJ, Roberts JM 1995 Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9:1149–1163[CrossRef][Medline]
  2. Chan FK, Zhang J, Cheng L, Shapiro DN, Winoto A 1995 Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4. Mol Cell Biol 15:2682–2688[Abstract]
  3. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B, Toyoshima H, Hunter T 1993 WAF1, a potential mediator of p53 tumor suppression p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 75:817–825[Medline]
  4. Guan KL, Jenkins CW, Li Y, Nichols MA, Wu X, O’Keefe CL, Matera AG, Xiong Y 1994 Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function. Genes Dev 8:2939–2952[Abstract]
  5. Hannon GJ, Beach D 1994 p15INK4B is a potential effector of TGF-ß-induced cell cycle arrest. Nature 371:257–261[CrossRef][Medline]
  6. Hirai H, Roussel MF, Kato JY, Ashmun RA, Sherr CJ 1995 Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. Mol Cell Biol 15:2672–2681[Abstract]
  7. Matsuoka S, Edwards MC, Bai C, Parker S, Zhang P, Baldini A, Harper JW, Elledge SJ 1995 p57KIP2, a structurally distinct member of the p21CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. Genes Dev 9:650–662[Abstract]
  8. Serrano M, Lee H, Chin L, Cordon-Cardo C, Beach D, DePinho RA 1996 Role of the INK4a locus in tumor suppression and cell mortality. Cell 85:27–37[Medline]
  9. Toyoshima H, Hunter T 1994 p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78:67–74[Medline]
  10. Sherr CJ, Roberts JM 1999 CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13:1501–1512[Free Full Text]
  11. Sicinski P, Donaher JL, Gene Y, Parker SB, Gardner H, Park MY, Robker RL, Richard JS, McGinnis LK, Biggers JD, Eppig JJ, Bronson RT, Elledge SJ, Weinberg RA 1996 Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature 384:470–474[CrossRef][Medline]
  12. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM 1996 A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85:733–744[Medline]
  13. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A 1996 Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27Kip1. Cell 85:721–732[Medline]
  14. Nakayama K, Ishida N, Shirane M, Inomata A, Inoue T, Shishido N, Horii I, Loh DY, Nakayama K 1996 Mice lacking p27(Kip1) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85:707–720[Medline]
  15. Cordon-Cardo C, Koff A, Drobnjak M, Capodieci P, Osman I, Millard SS, Gaudin PB, Fazzari M, Zhang ZF, Massague J, Scher HI 1998 Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma. J Natl Cancer Inst 90:1284–1291[Abstract/Free Full Text]
  16. Pierson TM, Matzuk MM, Transgenic mouse models to study the inhibins and activins. In: Muttukrishna S (ed) Inhibins, Activins, and Follistatin in Human Reproductive Physiology. Imperial College Press, London, in press
  17. Vale W, Bilezikjian LM, Rivier C 1994 Reproductive and other roles of inhibins and activins. In: Knobil E, Neill J (eds) The Physiology of Reproduction. Raven Press, Ltd., New York, pp 1861–1878
  18. Matzuk MM, Finegold MJ, Su J-GJ, Hsueh AJW, Bradley A 1992 {alpha}-Inhibin is a tumor-suppressor gene with gonadal specificity in mice. Nature 360:313–319[CrossRef][Medline]
  19. Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H, Bradley A 1994 Development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice. Proc Natl Acad Sci USA 91:8817–8821[Abstract]
  20. Langstein HN, Norton JA 1991 Mechanisms of cancer cachexia. Hematol Oncol Clin North Am 5:103–123[Medline]
  21. Oliff A, Defeo-Jones D, Boyer M, Martinez D, Kiefer D, Vuocolo G, Wolfe A, Socher SH 1987 Tumors secreting human TNF/cachectin induce cachexia in mice. Cell 50:555–563[Medline]
  22. Kumar TR, Palapattu G, Wang P, Woodruff TK, Boime I, Byrne MC, Matzuk MM 1999 Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis. Mol Endocrinol 13:851–865[Abstract/Free Full Text]
  23. Massague J 1990 The transforming growth factor-ß family. Annu Rev Cell Biol 6:597–641[CrossRef]
  24. Polyak K 1996 Negative regulation of cell growth by TGF ß. Biochim Biophys Acta 1242:185–199[Medline]
  25. Zauberman A, Oren M, Zipori D 1997 Involvement of p21(WAF1/Cip1), CDK4 and Rb in activin A mediated signaling leading to hepatoma cell growth inhibition. Oncogene 15:1705–1711[CrossRef][Medline]
  26. Yamato K, Koseki T, Ohguchi M, Kizaki M, Ikeda Y, Nishihara T 1997 Activin A induction of cell-cycle arrest involves modulation of cyclin D2 and p21CIP1/WAF1 in plasmacytic cells. Mol Endocrinol 11:1044–1052[Abstract/Free Full Text]
  27. Danila DC, Inder WJ, Zhang X, Alexander JM, Swearingen B, Hedley-Whyte ET, Klibanski A 2000 Activin effects on neoplastic proliferation of human pituitary tumors. J Clin Endocrinol Metab 85:1009–1015[Abstract/Free Full Text]
  28. Shikone T, Matzuk MM, Perlas E, Finegold MJ, Vale W, Bradley A, Hsueh AJW 1994 Characterization of gonadal tumor cell lines from inhibin-{alpha} deficient mice: the role of activin as an autocrine growth factor. Mol Endocrinol 8:983–995[Abstract]
  29. Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff A 1994 p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-ß and contact inhibition to cell cycle arrest. Genes Dev 8:9–22[Abstract]
  30. Beumer TL, Kiyokawa H, Roepers-Gajadien HL, Bos LAvd, Lock TM, Gademan IS, Rutgers DH, Koff A, Rooij DGd 1999 Regulatory role of p27kip1 in the mouse and human testis. Endocrinology 140:1834–1840[Abstract/Free Full Text]
  31. Tong W, Kiyokawa H, Soos TJ, Park MS, Soares VC, Manova K, Pollard JW, Koff A 1998 The absence of p27Kip1, an inhibitor of G1 cyclin-dependent kinases, uncouples differentiation and growth arrest during the granulosa -> luteal transition. Cell Growth Differ 9:787–794[Abstract]
  32. Boardman LA, Couch FJ, Burgart LJ, Schwartz D, Berry R, McDonnell SK, Schaid DJ, Hartmann LC, Schroeder JJ, Stratakis CA, Thibodeau SN 2000 Genetic heterogeneity in Peutz-Jeghers Syndrome. Hum Mutat 16:23–30[CrossRef][Medline]
  33. Matzuk MM, Kumar TR, Shou W, Coerver KA, Lau AL, Behringer RR, Finegold MJ 1996 Transgenic models to study the roles of inhibins and activins in reproduction, oncogenesis, and development. Recent Prog Horm Res 51:123–157[Medline]
  34. Coerver KA, Woodruff TK, Finegold MJ, Mather J, Bradley A, Matzuk MM 1996 Activin signaling through activin receptor type II causes the cachexia-like symptoms in inhibin-deficient mice. Mol Endocrinol 10:534–543[Abstract]
  35. Li Q, Karam SM, Coerver KA, Matzuk MM, Gordon JI 1998 Stimulation of activin receptor II signaling pathways inhibits differentiation of multiple gastric epithelial lineages. Mol Endocrinol 12:181–192[Abstract/Free Full Text]
  36. Loda M, Cukor B, Tam SW, Lavin P, Fiorentino M, Draetta GF, Jessup JM, Pagano M 1997 Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas. Nat Med 3:231–234[Medline]
  37. Porter PL, Malone KE, Heagerty PJ, Alexander GM, Gatti LA, Firpo EJ, Daling JR, Roberts JM 1997 Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients. Nat Med 3:222–225[Medline]
  38. Franklin DS, Godfrey VL, Lee H, Kovalev GI, Schoonhoven R, Chen-Kiang S, Su L, Xiong Y 1998 CDK inhibitors p18(INK4c) and p27(Kip1) mediate two separate pathways to collaboratively suppress pituitary tumorigenesis. Genes Dev 12:2899–2911[Abstract/Free Full Text]
  39. Lewis KA, Gray PC, Blount AL, MacConell LA, Wiater E, Bilezikjian LM, Vale W 2000 Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature 404:411–414[CrossRef][Medline]
  40. Chong H, Pangas SA, Bernard DJ, Wang E, Gitch J, Chen W, Draper LB, Cox ET, Woodruff TK 2000 Structure and expression of a membrane component of the inhibin receptor system. Endocrinology 141:2600–2607[Abstract/Free Full Text]
  41. Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, Korach KS 1999 Postnatal sex reversal of the ovaries in mice lacking estrogen receptors {alpha} and ß. Science 286:2328–2331[Abstract/Free Full Text]
  42. Rhee K, Wolgemuth DJ 1995 Cdk family genes are expressed not only in dividing but also in terminally differentiated mouse germ cells, suggesting their possible function during both cell division and differentiation. Dev Dyn 204:406–420[Medline]
  43. Pierson MP, DeMayo FJ, Matzuk MM, Tsai SY, O’Malley BW 2000 Regulable expression of inhibin A in wild-type and inhibin {alpha} null mice. Mol Endocrinol 14:1075–1085[Abstract/Free Full Text]
  44. Cipriano SC, Chen L, Kumar TR, Matzuk MM 2000 Follistatin is a modulator of gonadal tumor development in inhibin-deficient mice. Endocrinology 141:2319–2327[Abstract/Free Full Text]
  45. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM 1996 Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383:531–535[CrossRef][Medline]
  46. Cipriano SC, Chen YQ 1998 Insensitivity to growth inhibition by TGF-ß1 correlates with a lack of inhibition of the CDK2 activity in prostate carcinoma cells. Oncogene 17:1549–1556[CrossRef][Medline]