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Cell Growth & Differentiation Vol. 10, 785-796, December 1999
© 1999 American Association for Cancer Research

Dwarfism and Dysregulated Proliferation in Mice Overexpressing the MYC Antagonist MAD11

Christophe Quéva2, Grant A. McArthur3, Leni Sue Ramos and Robert N. Eisenman4

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The four members of the MAD family are bHLHZip proteins that heterodimerize with MAX and act as transcriptional repressors. The switch from MYC-MAX complexes to MAD-MAX complexes has been postulated to couple cell-cycle arrest with differentiation. The ectopic expression of Mad1 in transgenic mice led to early postnatal lethality and dwarfism and had a profound inhibitory effect on the proliferation of the hematopoietic cells and embryonic fibroblasts derived from these animals. Compared to wild-type cells, Mad1 transgenic fibroblasts arrested with altered morphology and reduced density at confluence, cycled more slowly, and were delayed in their progression from G0 to the S phase. These changes were accompanied by accumulation of hypophosphorylated retinoblastoma protein and p130. Cyclin D1-associated kinase activity was dramatically reduced in MAD1-overexpressing fibroblasts. However, wild-type cell-cycle distribution and morphology could be rescued in the Mad1 transgenic cells by the introduction of HPV-E7, but not an E7 mutant incapable of binding to pocket proteins. This indicates that the activities of the retinoblastoma family members, via the cyclin D pathway, are likely to be the major targets for MAD1-mediated inhibition of proliferation in primary mouse fibroblasts.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The control of cell number is crucial for ensuring an orderly deposition of tissues during embryonic development and, later in life, for homeostasis and a regulated response to stress (1) . That disruption of cell number regulation has important consequences was underscored by recent reports of the inactivation in mice of the CDKI5 p27KIP1. Loss of p27KIP1 results in hypercellularity of all tissues, as well as excessive proliferation in response to inflammation (2, 3, 4, 5) . This type of deregulation is also a prelude to tumorigenesis (6 , 7) .

Direct regulation of the cell-cycle machinery itself, as for example through the CDKIs, has been definitively shown to be of major importance in the control of cell number (1 , 6 , 7) . More recently, it has also become apparent that transcriptional regulation plays a fundamental role in the regulation of cell number. This is best exemplified by the pRb/E2F pathway in the regulation of G1 to S phase progression in the cell cycle. Here, phosphorylation of Rb by the G1 cyclin-dependent kinases leads to the dissociation of Rb from E2F, followed by derepression and activation of E2F target genes required for entry in S (8, 9, 10, 11) .

The MYC oncoproteins, encoded by the c-, N-, L-, and s-Myc genes and their antagonists, the MAD protein family (MAD1, MXI1, MAD3 and MAD4), are involved in the control of cell growth, apoptosis, cell-cycle progression, and cell-cycle exit upon differentiation (see Ref. 12 for review). The MYC and MAD families, as well as the recently identified MNT (ROX) protein, are transcription factors that independently heterodimerize with the widely expressed, stable, adapter protein MAX (13, 14, 15, 16, 17, 18) . The different MAX-containing heterocomplexes are all capable of binding to the same E-box (CACGTG) DNA sequence in vitro as well as more distantly related noncanonical sites both in vitro and in vivo (see Ref. 19 for review). MYC-MAX heterodimers activate transcription, although there is also evidence for a repression function associated with distinct binding sites (20, 21, 22, 23) . MAD-MAX and MNT-MAX are known to repress transcription at E-box binding sites primarily through the recruitment of mSin3-histone deacetylase corepressor complexes (14, 15, 16, 17, 18 , 24 , 25) . It has been proposed that histone deacetylase-mediated removal of the acetyl groups on the N-terminal tails of histones H3 and H4 induces the formation of a repressive chromatin structure and is the basis of MAD- and MNT-mediated repression (for reviews, see Refs. 26, 27, 28, 29) .

It has been suggested that the specific transcriptional response mediated by the MAX network is dependent on the relative abundance of the different MAX partners: MYC, MAD, and MNT. Expression of the Myc family genes is confined primarily to proliferating cells (12 , 30) . c-Myc is induced rapidly in response to mitogenic stimuli and is down-regulated upon cell-cycle exit and differentiation. Several lines of evidence suggest that the induction of c-Myc is a rate-limiting step during cell-cycle entry and the G1-S transition. For example, the activation of c-Myc is sufficient to induce cell-cycle entry in some lines of serum-starved fibroblasts (31) , and its expression is required for progression in response to mitogenic stimulation (32) . Moreover, constitutive expression of c-Myc in cell lines and in transgenic mice attenuates cell-cycle withdrawal and terminal differentiation and induces apoptosis in the absence of survival factors (33, 34, 35, 36) . Conversely, inactivation of c-Myc prevents G1-S transition (37 , 38) , and targeted disruptions of c-Myc or N-Myc in mice result in midgestational lethality accompanied by a failure of cell proliferation during organogenesis (see Ref. 39 for review).

In contrast to Myc, Mad gene expression is associated with cell-cycle exit prior to differentiation (15 , 18 , 30 , 40, 41, 42, 43, 44, 45, 46, 47) . With the exception of Mad3, which is expressed transiently during the last cell cycle preceding growth arrest, Mxi1, Mad4, and Mad1 transcripts are strongly induced in postmitotic cells in the developing mouse embryo (15 , 30) . In myeloid cells Mad1 and Mxi1, mRNAs are detected as "immediate early" responses to differentiation inducers. Differentiation of these cells as well as keratinocytes is accompanied by a switch from MYC-MAX complexes to MAD1-MAX complexes that are thought to down-modulate genes important for cell-cycle progression (40 , 41) . Indeed, overexpression of MAD1 and MXI1 in cell lines inhibits cell proliferation by promoting G1 arrest. MAD1 and MXI1 overexpression has also been shown to suppress transformation (46 , 48, 49, 50, 51, 52, 53) . Conversely, targeted disruption of Mad1 in mice demonstrated a function for Mad1 in facilitating the cell-cycle exit of late granulocytic precursors, although these cells do differentiate (54) . Analysis of Mxi1 deficient mice also indicated an increased fraction of cycling cells in a variety of tissues as well as an augmented susceptibility to tumorigenesis, which is consistent with the notion that Mad1 and Mxi1 have related but distinct roles in promoting cell-cycle exit (55) .

The targeted disruption of Mad1 in mice appeared to affect only a limited number of cell types, and in vitro assays were required to detect a phenotype (54) . Homozygous deletion of Mxi1 influenced more cell types, but the hyperproliferation and oncogenic effects were only detected in aging animals (55) . In addition, the proliferation inhibitory response to Mad1 and Mxi1 overexpression were tested only in cell lines and some tumors (47 , 49 , 52 , 53) . To determine whether Mad1 can influence the functions of multiple cell types and also affect normal development, we generated transgenic mice ubiquitously overexpressing Mad1. We describe here the effect of ectopic Mad1 expression in mice and on the cell cycle in the primary fibroblasts derived from these mice.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Neonatal Lethality in BAP-Mad1 Transgenic Mice.
To investigate the effect of Mad1 during development, we have expressed the Mad1 cDNA ubiquitously in mice using the human BAP (BAP-Mad1; Ref. 56 ). Because it has been reported to be difficult to achieve high stable expression of Mad1 in cell lines (see, for example, Ref. 47 ), we were initially surprised to obtain founder animals. However, the percentage of transgenic animals following pronuclear injection was low. Only 8% (6 of 71) of the F0 animals had integrated the BAP-Mad1 DNA. Among these, only two founder mice were able to transmit the transgene to further generations.

F1 animals heterozygous for the BAP-Mad1 allele were bred to wild-type C57BL/6J females. Using in situ hybridization (Fig. 1)Citation and RT-PCR to detect Mad1 expression at day 10.5 post coitus, we first documented overexpression of Mad1 in all embryonic derivatives of BAP-Mad1 transgenic embryos. MAD1 protein was also up-regulated in embryonic fibroblasts harvested from the transgenic mice (Fig. 5A)Citation . In adults, RT-PCR analysis demonstrated increased expression of Mad1 in a subset of organs, albeit at a lower level than in the embryo6 . We then analyzed the distribution of the BAP-Mad1 transgene in these crosses. We found that, at weaning age, the BAP-Mad1 transgenic animals were significantly underrepresented, with 82% below the expected Mendelian frequency in the B3 line and 67% in the B2 line (Table 1)Citation . To establish the time of death of the transgenic mice, we collected embryos at different stages during development. Table 2Citation indicates that the BAP-Mad1 pups at 13.5 p.c. and at 18.5 p.c. were recovered with the expected frequency in crosses between heterozygous transgenic males and wild-type females. Because no major abnormalities could be observed in the 18.5-p.c. embryos, when development is complete, the deficit observed in the number of transgenic mice is likely to be due to lethality during the first week of life. Because of the difficulty in obtaining transgenic pups, we have not investigated the cause of death in greater detail. However, careful observation of newborn litters revealed that some of the newborn mice did not establish normal feeding and died within the first 24 h after birth. Because no death was observed beyond this initial period of 24 h, we concluded that expression of Mad1 under the BAP leads to perinatal lethality with partial penetrance. The mice that died were clearly growth retarded compared to their littermates. The transgenic embryos at 18.5 p.c. weighed on average 22% less (transgenic: 1.14 g ± 0.13; wild-type: 1.47 g ± 0.05; P < 0.001). The dwarfism of BAP-Mad1 embryos was already evident at 12.5 p.c6. These embryos were morphologically normal, except for the edges of the truncal neural tube that were consistently more widely separated compared to their siblings. No difference in the BrdUrd staining of S-phase cells was visible in the neural tube or in the whole embryo.6 The expression of the other Mad or Myc genes was not significantly affected in the BAP-Mad1 transgenic mice at 11.5-p.c. days of development.6 In conclusion, Mad1 overexpression driven by the BAP results in reduced size and weight but does not produce evident morphogenetic abnormalities.



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Fig. 1. Expression of Mad1 in BAP-Mad1 transgenic mice. Transverse section through a wild-type (A) and a BAP-Mad1 transgenic embryo (B) at 10.5 days of development hybridized with a Mad1-specific riboprobe. The signal obtained in the transgenic embryo is clearly more intense all over this section, demonstrating ubiquitous expression of the transgene.

 


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Fig. 5. Molecular analysis of cell-cycle regulators in BAP-Mad1 MEFs. A, Western blot analysis of the regulators of G1 phase progression. Equal amounts of proteins prepared from exponentially growing MEFs at 50% confluence were separated by SDS-PAGE and transferred on polyvinylidene difluoride membranes. The cellular proteins visualized in each panel are indicated on the left. Two bands migrating to the apparent molecular weight of Mr 68,000 and Mr 75,000 were detected for CDC25a; the addition of phosphatase to the samples provoked the collapse of the two bands into the faster migrating form.6 B, Mad1 overexpression leads to a decrease in CDK2 kinase activity without changing cyclin E1- and cyclin A-associated kinase activity. Cyclins D1, A, and E and CDK4 and CDK2 were immunoprecipitated from equal amounts of protein extracts from MEFs derived from individual embryos, wild-type (wt), or transgenic (Tg). Kinase activity in the immunoprecipitates was assayed in vitro on exogenously added substrates: GST-Rb for cyclin D1 and CDK4 and histone H1 for cyclin E1, cyclin A, and CDK2.

 

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Table 1 Frequency of BAP-Mad1 transgenic mice in crosses between F1 animals heterozygous for the BAP-Mad1 allele and wild-type C57BL/6J 10 days after birtha

 

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Table 2 Frequency of BAP-Mad1 transgenic embryos at 12.5–13.5 p.c. and 18.5 p.c. in crosses between F1 animals heterozygous for the BAP-Mad1 allele and wild-type C57BL/6J.a

 
Phenotype of the Surviving Mad1 Transgenic Mice.
All of the surviving BAP-Mad1 transgenic mice displayed a similar phenotype. They were consistently smaller than their nontransgenic littermates, allowing them to be easily distinguished in a litter (Fig. 2A)Citation . The dwarfism of BAP-Mad1 transgenic animals was apparent in each of the founder lines, suggesting that the phenotype was not the result of a specific integration site. The weight loss was 17% (P < 0.02 calculated for males and females separately at 3 weeks of age) and 23% (P < 0.02 calculated for males and females separately at 3 weeks of age) in the B2 and B3 lines, respectively, and it was proportionally identical for males and females. Because the B3 line was more severely affected by the loss of weight and by mortality than the B2 line, we concentrated on the progeny of the B3 founder; however, the major phenotypes observed in the B2 line (see below) were confirmed for the B3 line as well. Fig. 2BCitation shows a representative growth curve of two transgenic males compared to sex-matched siblings. Dwarfism was apparent at birth and was sustained throughout adult life of the BAP-Mad1 mice (monitored for up to 1 year). In addition to their decreased size and weight, the BAP-Mad1 mice displayed delayed onset of cutaneous pigmentation, fur growth, and eye opening during the neonatal period. Otherwise, the transgenic animals were morphologically and behaviorally normal. No illnesses were observed over a 12-month period. At necropsy, no apparent histological abnormalities were detected in any organs, which had weights proportional to the total body weight (Fig. 2C)Citation . A notable exception was the brain whose weight was identical in wild-type and transgenic mice, despite a 3-fold-increase in Mad1 mRNA expression in this organ, as estimated by RT-PCR6. Similarly, ectopic expression of Mad1 in the neural stem cells obtained in mice transgenic for a rat Nestin 2nd intron-TK-Mad1 construct did not lead to any neurological phenotypes.6



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Fig. 2. A, representative picture of 6-day-old pups showing the growth deficit of the BAP-Mad1 transgenic embryos (TG; center) compared to wild-type (WT) littermates (periphery). B, individual growth curves of all transgenic (Tg; {blacksquare}) and nontransgenic (wt; {square}) male siblings derived from a single litter. Mice overexpressing Mad1 had bodyweights 20% less than littermate control, starting from birth and continuing through adult life. C, mean of organ weights from six control and six BAP-Mad1 transgenic male mice at 8 weeks of age. Plotted is the percent decrease in weight in the transgenic mice (Tg; ) compared with the wild-type siblings (wt; {blacksquare}; set to 100).

 
The possibility that the dwarfism detected in the Mad1 transgenic animals was due to abnormal secretion of GHs was evaluated by measuring the serum levels of IGF-I and GH in 4–8-week-old adults. The mean serum levels of these major determinants of growth in at least four BAP-Mad1 transgenic animals were not significantly different from levels in control littermates. The concentration of GH was 77 ± 107 ng/ml in wild-type mice and 28 ± 24 ng/ml in the Mad1 transgenics. For IGF-I, whose excretion is under the control of GH, the concentration was 409 ± 109 ng/ml in wild-type and 400 ± 77 ng/ml in transgenic mice. Cell densities were determined for the embryonic thymus, the hip cartilage, and the adult liver by counting nuclei in a fixed volume of an anatomically identical region. No significant differences were observed (for example, the number of hepatocytes per unit of volume was 151 ± 20 in wild-type and 133 ± 34 in transgenic mice). This result suggested that a reduced total cell number per organ and not reduced cell size is responsible for the reduced organ size in BAP-Mad1 mice. This conclusion was supported by the similar size of MEFs and lymphoid and myeloid cells from wild-type and transgenic mice as assessed by flow cytometry.6 In addition, the absence of a significant difference in IGF-I and GH levels raised the possibility that the effects of Mad1 on cell number were cell autonomous. To investigate this possibility in more detail, we derived hematopoietic and fibroblast cells from the wild-type and Mad1 transgenic mice and studied their properties in vitro.

Reduced Proliferative Potential in Mad1-expressing Hematopoietic Progenitors.
To examine the effect of Mad1 on the proliferation of hematopoietic cells, we used in vitro colony assays as a measure of the frequency of specific bone-marrow precursor cells, as well as their capacity to generate differentiated progeny (see Ref. 57 for review). Bone marrow cultures were stimulated with recombinant cytokines to generate both myeloid and erythroid colonies. Colonies obtained from cultures of cells from BAP-Mad1 transgenic mice were both less numerous (Fig. 3A)Citation and composed of fewer cells (Fig. 3B)Citation compared to cultures from littermate controls. Mad1 had similar inhibitory effects on myeloid cell (CFU-G, CFU-GM, and CFU-M) and erythroid cell proliferation (BFU-E and CFU-E). Examination of the composition of myeloid colonies from fixed cultures did not reveal any significant morphological difference between transgenic and normal littermate controls.



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Fig. 3. Reduced numbers and proliferation of colony-forming cells from BAP-Mad1 transgenic mice. Bone marrow cells were cultured in semisolid media at 25,000 cells/ml and 50,000 cells/ml for analysis of nonerythroid colonies and erythroid colonies, respectively. Colonies were stimulated with the following cytokines: BFU-E: IL-3 plus Epo; CFU-E: Epo; CFU-G: granulocyte colony-stimulating factor; CFU-GM: GM-CSF; and CFU-M: macrophage colony stimulating factor. A, colony numbers were counted after 7 days in culture for CFU-G, CFU-GM, and CFU-M; 3 days for CFU-E; and 8 days for BFU-E. Data shown are the total number of colonies per femur. Data corrected for total bone marrow cellularity were similar because BAP-Mad1 transgenic mice had a reduction in bone marrow cellularity proportional to the reduction in total body weight ({approx}20%). P < 0.05 for numbers of colonies in cultures from BAP-Mad1 transgenic mice compared to littermate controls. B, numbers of cells in each colony (defined as >50 cells/clone) were determined by microscopy of fixed myeloid cultures (a minimum of 14 colonies were analyzed for each stimulus). P < 0.05 for CFU-G and CFU-GM from BAP-Mad1 transgenic mice compared to littermate controls. C, the survival of CFU-GM in cultures deprived of cytokines was determined by culturing bone marrow cells for an initial period without the addition of any cytokines. Twenty-four or 48 h after the initiation of the culture, cells were stimulated with GM-CSF. The number of colonies was determined 7 days later and compared to numbers in cultures stimulated with GM-CSF without a period of cytokine deprivation.

 
The reduction in numbers of colony-forming cells was not accompanied by a proportional reduction in the numbers of mature hematopoietic cells in the bone marrow or spleen.6 Because c-Myc (34 , 58) and Mad1 (54) have been implicated in the regulation of apoptosis, particularly following withdrawal of survival factors, we examined the effect of cytokine withdrawal on the ability of colony-forming cells to survive and proliferate in vitro. Colony-forming cells from BAP-Mad1 transgenic mice displayed enhanced survival in experiments in which the addition of GM-CSF was delayed for 24 or 48 h after the initiation of the culture (Fig. 3C)Citation . These results are consistent with a reduced apoptotic response of myeloid precursor cells from BAP-Mad1 transgenic mice when cultured under conditions where survival factor is limiting.

Altered Proliferation of Fibroblasts Derived from Mad1 Transgenic Embryos.
The earlier observation that ectopic expression of Mad1 inhibited the proliferative response of NIH-3T3 fibroblasts to signaling though the colony-stimulating factor-1 receptor (52) prompted us to study the growth properties of primary MEFs derived from BAP-Mad1 transgenic embryos. Fig. 4ACitation shows that the proliferation rates and the saturation densities were strikingly different depending on whether the MEF cultures were derived from wild-type or BAP-Mad1 transgenic embryos. MEFs overexpressing the MAD1 protein (see the Western blot in Fig. 5ACitation ) showed altered proliferative capacity with a doubling time of 80 h, compared to 24 h for the wild-type MEFs. FACS analysis of exponentially growing cells obtained during the second day of the growth rate study revealed a significant increase in the fraction of cells in the G1 phase and a concomitant decrease in the S phase in MEFs derived from the Mad1 transgenic embryos compared to the wild-type (Fig. 4B)Citation MEFs. A decrease was also apparent in the G2-M phase fraction, but this was more difficult to quantify accurately because of contamination of the 4N DNA fraction with tetraploid cells in G1. Similar results were obtained by measuring BrdUrd incorporation in two wild-type and two transgenic MEF cultures derived from the B2 founder line.6 Furthermore, re-entry into the cell cycle after arrest in G0 following serum starvation was delayed in MEFs derived from the transgenic embryos (Fig. 4C)Citation . Arrest after 30 h in 0.1% FCS was nearly complete for both the transgenic (%S = 3.7 ± 0.7) and wild-type cells (%S = 4.0 ± 0.1). Following stimulation with medium containing 10% serum, wild-type MEFs began to enter S at 10–14 h. In contrast, a significant increase in the S phase fraction of the Mad1 MEFs could only be detected at 17 h.



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Fig. 4. A proliferation defect in early-passage MEFs derived from BAP-Mad1 transgenic mice. A, growth curve of MEFs derived from three wild-type (solid line) and 3 BAP-Mad1 (dashed line) transgenic littermate embryos. Six-centimeter dishes were seeded with 2 x 105 cells and counted after the indicated days. B, analysis of cell-cycle stages in exponentially growing cultures of wild-type (wt) and BAP-Mad1 (Tg) MEF cultures. Plotted is the average proportion of cells in G1 and in S for three independent cultures as determined by FACS. C, kinetics of S-phase entry of MEFs derived from BAP-Mad1 embryos and nontransgenic siblings after arrest in G0 obtained by serum withdrawal. Asynchronous cultures were placed in DMEM containing 0.1% FBS for 30 h. They were released from G0 block by replacing the media with DMEM, 10% FBS and analyzed by FACS. Plotted is the percentage of cells in the S phase of the cell cycle as a function of time in hours. Cell numbers are as they are in A. The MEFs expressing MAD1 entered the S phase with a 3-h delay compared to the wild-type.

 
The density at saturation of the Mad1 MEFs (mean for the three lines, 9 x 103 ± 2 cells/cm2) was also strikingly lower compared to the wild-type MEFs (mean, 23 x 103 ± 6 cells/cm2; Fig. 4ACitation ). Forward scatter analysis by FACS of the MEFs did not show an altered size for the MEFs expressing Mad1 compared to the wild-type6 MEFs, suggesting that Mad1 MEFs do not have an increased volume but may rather spread over a wider area than the wild-type MEFs. This phenomena was apparent in the morphological differences between the cultures (Fig. 6B)Citation , LXSN-infected cells). Wild-type MEFs appeared small and refringent. By contrast, Mad1 MEF cultures were occupied by flattened cells. We hypothesized that the Mad1-overexpressing cells were more sensitive to the growth inhibitory signals produced in subconfluent cultures and hence, arrested at a lower density.



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Fig. 6. HPV-16E7 protein, but not a mutant defective in Rb binding, reverses the quiescent phenotype of MEFs from BAP-Mad1 transgenic mice. A, MEFs were infected with the indicated retroviruses and selected in G418. Seven days after infection, cells were analyzed for DNA content by flow cytometry. Percentages were calculated by gating on diploid cells in the G0/G1 and S phases of the cell cycle and excluding cells with a 4N DNA content because of the high frequency of tetraploid cells in cultures from BAP-Mad1 transgenic mice. B, morphology of MEFs 7 days after retroviral infection. Note an increase in refractory cells in cultures of BAP-Mad1 fibroblasts infected with the 16E7-encoding retrovirus compared to BAP-Mad1 fibroblasts infected with control retroviruses.

 
In conclusion, the MEFs derived from the BAP-Mad1 mice possess a decreased proliferative capacity, accumulate in the G0/G1 phase, and possibly have an increased sensitivity to contact inhibition. We next investigated the molecular mechanism underlying this phenotype.

CDK Activities Are Targets of MAD1.
The cell-cycle phenotype of the fibroblasts derived from the BAP-Mad1 transgenic embryos was first investigated by Western blot analysis of the levels of major cell-cycle regulatory proteins. To avoid bias in the results due to the difference in cell density (see above), extracts equalized for protein content were prepared from exponentially growing cells at 50% confluence both for wild-type and transgenic MEFs. We focused our analysis on the regulators of the G1 to S transition because the activation of the conditional alleles of MYC in quiescent fibroblasts had been previously shown to promote the rapid activation of G1 cyclin-dependent kinases (59 , 60) . Western blot analysis of our MEF cultures showed that very low levels of MAD1 could be detected in wild-type MEFs and that there was an {approx}3-fold increase in MAD1 expression in the BAP-Mad1 transgenic cells. No changes in the levels of c-Myc were observed.6 We also detected a slight up-regulation of the CDKIs p21Cip1 and p27Kip1, whereas the amount of the CDC25a phosphatase, reported to be a MYC-target gene (61) , was slightly reduced in the transgenic embryo-derived fibroblasts (Fig. 5A)Citation . In wild-type exponentially growing MEFs, pRb was present as a diffuse band (the upper band at 110 kDa in Fig. 5ACitation ) indicative of hyperphosphorylated forms, as expected in a population of proliferating cells. By contrast, in Mad1 MEFs, virtually all pRb proteins were detected as a faster migrating, hypophosphorylated form (Fig. 5A)Citation .

More striking differences between wild-type and transgenic MEFs were observed in the level of expression of the Rb-related protein p130 and, to a lesser extent, p107 (Fig. 5A)Citation . p107 is usually found to be associated with E2F4 in cycling cells in the late G1 and S phases (see Refs. 8, 9, 10, 11 for reviews). By contrast, p130 is detected primarily in quiescent cells and is degraded upon cell-cycle entry (62) . As expected, p107 levels were reduced in Mad1 MEFs, which showed a decrease in the proportion of cells in the S phase. Whereas p130 was in very low abundance in wild-type MEFs (Fig. 5A)Citation , the fibroblasts harvested from the transgenic mice accumulated p130, a surprising result for cells that were proliferating exponentially (Fig. 4A)Citation . These data demonstrate that increased expression of MAD1 in proliferating fibroblasts leads to the accumulation of p130 and hypophosphorylated Rb as would normally be observed in quiescent cells.

We next tested whether the altered growth and Rb phosphorylation phenotype could be caused by changes in cyclin or CDK expression. Western blot analysis of the same extracts showed that no significant changes could be observed in the amounts of the cyclins and CDKs known to play a role in G1 to S progression (Fig. 5A)Citation . Surprisingly, measurement of the histone H1 kinase activity associated with cyclin E1 and cyclin A did not reveal any decrease due to MAD1 overexpression (Fig. 5B)Citation . This result was also confirmed in MEFs derived from the B2 transgenic mice.6 Similar experiments conducted on immunoprecipitates of the catalytic moiety of the cyclin E1 and A complexes, namely CDK2, consistently show an average 30% decrease in the phosphorylation of histone H1 (Fig. 5B)Citation . This result suggests that MAD1 influences CDK2 when CDK2 is not associated with cyclins E1 or A.

Cyclin D1 and its kinase partners CDK4 and CDK6 also participate in the phosphorylation of pRb and p130 in G1. In MEFs derived from BAP-Mad1 transgenic embryos, the kinase activity of CDK4, as well as the cyclin D1-associated kinase activity, were dramatically reduced (Fig. 5B)Citation . Quantitation of 32P incorporation in GST-pRb by phosphorimager (Molecular Dynamics) showed a 60% average decrease. These data demonstrate that cyclin D1/CDK4 and the pRb family are significantly affected by MAD1 expression in fibroblasts.

HPV-16E7 Rescues the MAD1 Overexpression Phenotype.
MEFs from BAP-Mad1 transgenic mice displayed reduced cyclin D1-associated kinase activity, which was accompanied by Rb hypophosphorylation and the accumulation of another Rb family member, p130. To test whether the decreased proliferation rate of BAP-Mad1 MEFs was dependent on members of the Rb family, we took advantage of the properties of the HPV-E7 protein. The oncogenic HPV-16E7 protein induces cellular DNA synthesis at least in part through direct protein-protein interactions that target a series of key cellular regulators, including pRb, p107 and p130, and p21Cip1 and p27Kip1, thereby relieving the requirement for cyclin D1 function in G1 (8 , 11) . HPV-16E7(24G) contains an amino acid substitution at residue 24 in HPV-16E7, which prevents it from binding to pRb, p107, and p130 and therefore, dissociates the inhibitory effects of E7 on CDKIs from its effects on the pRb family7 . We therefore used retrovirus-mediated gene transfer to express both full-length HPV-16E7 and HPV-16E7(24G) (carrying an amino acid substitution at residue 24) in MEFs from wild-type and BAP-Mad1 transgenic mice.

We found that expression of HPV-16E7 in MEFs rescued the phenotype induced by Mad1 overexpression. HPV-16E7 increased the S-phase fraction of diploid MEFs from BAP-Mad1 transgenic mice to a similar level to that observed in wild-type cells infected with a control retrovirus (Fig. 6A)Citation . In contrast, cells derived from the BAP-Mad1 transgenic mice had a similar diploid S-phase fraction when infected with a retrovirus encoding the HPV-16E7(24G) molecule or with the control LXSN retrovirus. Furthermore, the expression of HPV-16E7, but not HPV-E7(24G), reversed the flattened morphology of MEFs from BAP-Mad1 transgenic mice (Fig. 6B)Citation . These findings support the idea that a crucial aspect of the growth and morphological phenotypes of the BAP-Mad1 transgenic MEFs is an inability to inactivate the Rb family of proteins.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Studies on the properties and interactions among members of the Max network have suggested that a balance between MYC-MAX and MAD-MAX and MNT-MAX heterodimers may be an important regulator of cell proliferation and differentiation (28 , 63) . Ectopic expression of Mad1 or Mxi1 promotes arrest in G1 (49 , 52) , and cell lines stably overexpressing Mad1 have been difficult to obtain. Given that the inactivation of c-Myc or N-Myc (39) and the overexpression of Mnt using the human BAP (16) leads to early embryonic lethality, we were somewhat surprised to obtain mice transgenic for BAP-Mad1.

The most striking developmental phenotypes of the two BAP-Mad1 transgenic lines are dwarfism and perinatal lethality. Transgenic embryos from 12.5 p.c. were smaller, albeit morphologically normal, and development proceeded normally until birth. However, up to 80% of the transgenic pups died during the first postnatal week. Because the survivors weighed 20% less than wild-type mice, we hypothesize that the lethality itself is due to the more severely retarded pups being too immature to survive. Many mutations in mice have been reported to produce dwarfism. In general, these fall into two categories. First, those that perturb the endocrine control of growth in the organism, as for example, due to the loss of insulin-like growth factor, GH, or the pituitary somatotroph cells (64, 65, 66) . These mutations would not be expected to be cell-autonomous. Second, dwarfism has been reported in cases of overexpression of negative regulators of the cell cycle, such as Rb (67) , or of inactivation of positive effectors of the cell cycle, such as cyclin D1 (68 , 69) . These genes are likely to affect growth in a cell-autonomous matter. In our BAP-Mad1 mice, no significant differences could be detected in the blood concentrations of IGF-I or GH compared to wild-type littermates. In addition, we were able to demonstrate that embryonic fibroblasts, as well as myeloid and erythroid cells isolated from the transgenic mice, were not able to proliferate at rates comparable to wild-type cells when stimulated in vitro with maximal concentrations of growth factors (see below). It is therefore likely that the dwarfism affecting the BAP-Mad1 transgenic mice is caused by cell autonomous hypoproliferation.

Another aspect of the BAP-Mad1 phenotype is the apparent disparity between in vivo and in vitro effects on cell growth. In vitro, the population doubling time of the BAP-Mad1 MEF cultures was increased by 3-fold, and the fraction of cells in the S phase was reduced by 30%. Nevertheless, in vivo, the pattern of cells that incorporated BrdUrd was similar in wild-type and transgenic embryos.6 However, a moderate reduction in the number of BrdUrd-positive cells, while being very difficult to detect at any given time in an embryo, could potentially have a significant effect on the size of the animal. Similarly, the phenotype displayed by hematopoietic progenitors was much more severe in vitro than in vivo. The number and the frequency of the different circulating hematopoietic cells were not significantly different between wild-type and transgenic animals.6 However, when placed in culture, the number of erythroid and myeloid progenitors was reduced up to 5-fold when derived from the transgenic animals. The BAP-Mad1 mice must therefore possess compensatory mechanisms to adapt to Mad1 overexpression. One possible mechanism is that the cell-number deficit is compensated by decreased cell death. Indeed, we found that the BAP-Mad1 hematopoietic cells were less sensitive to apoptosis induced by growth-factor withdrawal. Interestingly the opposite effect, i.e., increased apoptosis, had been previously indicated in homozygous Mad1 null mutant mice, whereas the increased numbers of late myeloid precursors detected in bone marrow culture were found to be more sensitive to cell death upon withdrawal of survival factors (54) . Augmented sensitivity to apoptosis may account for the near normal numbers of mature myeloid cells in the Mad1-/- mice. However the protection against cell death by elevated Mad1 levels is not absolute because wild-type or transgenic-derived cells were equally sensitive to apoptosis by radiation or staurosporine treatment6.

Mad1 transgenic mice defective in cell proliferation provide unique tools to investigate the molecular mechanisms of the action of MAD1 on the cell cycle in primary cells. Because the MEFs derived from the BAP-Mad1 transgenic embryos accumulated in the G0/G1 phase of the cell cycle, we focused our analysis on known regulators of G1 progression and cyclin D/CDK4 and cyclin E1/CDK2 complexes. Assay of the kinase activity associated with cyclin 1E and A did not reveal any differences between the wild-type fibroblasts and those overexpressing Mad1. CDK2 activity, when assayed independently, was decreased by 30% in the transgenic fibroblasts. This result suggested that MAD1 may reduce CDK2 activity by means independent of the cyclins E1 and A. One possibility may be the presence in fibroblasts of a yet unknown cyclin that is regulated by MAD1 and associated with CDK2. The recently described cyclin E2 would be a good potential candidate for such activities (70, 71, 72) . The major effect of MAD1 overexpression in fibroblasts is on cyclin D1/CDK4 kinase activity, which was reduced by 60%. A direct consequence of the decreased kinase activity of the cyclin D1/CDK4 complexes was the accumulation of hypophosphorylated pRb and of the Rb-related protein p130. p130 is normally detected in quiescent cells and is degraded following phosphorylation by CDK4 and cell-cycle entry (62) . The introduction of the oncogenic HPV-16E7 protein led to a conversion of both the morphology and cell-cycle profile of the Mad1 MEFs toward wild-type. Because the conversion depended on the ability of HPV16-E7 to inhibit Rb family proteins, these findings taken together suggest that Mad1 acts during the cell cycle mainly through the cyclin D-Rb/p130 pathway. This finding is consistent with a previous study demonstrating that Mad1 and Mxi1 fail to inhibit cotransformation by E1a and Ras, thus indicating that Mad acts upstream of Rb (51) .

The reason underlying the effect of MAD1 on cyclin D1/CDK4 kinase activity was investigated by looking at some known regulators of this complex. Although our attempts to detect the INK4 family proteins, which specifically inhibit CDK4 and CDK6, were unsuccessful, we did observe increased expression of the CDKIs p21Waf1 and p27Kip1, which could, in principle, be responsible for the negative effect on cell proliferation. Another potential target of MAD1 repression could be the tyrosine phosphatase CDC25a, which has been shown to be up-regulated by c-MYC (61) and down-regulated in a Mad1-overexpressing cell line (73) . Indeed, we detect a slight but consistent decrease in the amount of CDC25a in the MEFs derived from the BAP-Mad1 transgenic mice. Interestingly, in vivo in cells missing p15 INK4B, transforming growth factor ß can trigger cell cycle arrest by inhibiting the expression of CDC25a. This leads to the accumulation of tyrosine phosphorylated forms of CDK4 and CDK6 but not CDK2, and specific inhibition of cyclin D-associated kinase activity (74) . It will be interesting to test whether a Mad protein is directly responsible for the repression of CDC25a by transforming growth factor ß.

The specific effect of MAD1 on the cyclin D pathway is surprising and bears on the relationship between MYC and MAD. There is extensive literature on MYC inducing a rapid up-regulation of cyclin E1-associated kinase activity and also cyclin A expression (59 , 75, 76, 77, 78, 79) . Induction of MYC activity in Rat1-MycER cells leads also to a slower induction of cyclin D1-associated kinase activity without change in cyclin D1 expression (59) . A recent study (80) has shown that homozygous inactivation of c-Myc in a fibroblast cell line led to a 12-fold decrease in cyclin D1/CDK4,6 activities compared to c-Myc+/+ cells. Cyclin E/CDK2 activity was also delayed and reduced in amplitude but to a lesser extent. Interestingly, the slow growth rate of the c-Myc-/- cells could not be corrected by the restoration of CDK4 and CDK6 activity, showing that MYC affects multiple aspects of cell division. The more prominent defect in MAD1-overexpressing MEFs was also a reduction in cyclin D-associated kinase activity. One interpretation is that MAD and MYC targets influence cyclin D and E pathways depending on the cellular context. Our experiments were performed in primary cultures of embryonic fibroblasts, whereas the MYC effect on cyclin E1 was demonstrated in the Rat-1 and NIH3T3 cell lines (59 , 75, 76, 77, 78, 79) . An alternative interpretation is that MAD1 was actually ineffective in repressing the MEF genes normally regulated by MYC, which mediate the activation of the cyclin E1 pathway and inactivation of p27KIP1. Such hypotheses are difficult to reconcile given our present knowledge of these molecules. Indeed, MYC and MAD proteins are highly homologous in the basic region, recognize the same E-box sequences in vitro, and were thought to have similar targets in vivo. ODC, a well characterized target of MYC, is repressed by MXI1 (81) and by MAD18 in transient expression experiments. However, a specific promoter context can discriminate between MYC and the E-box binding bHLHZip USF and TFE3 (82 , 83) . Such interactions occurring at the regulatory sequences of genes lying in the cyclin E1 pathway could be responsible for their apparent protection against MAD-mediated transcriptional repression. Furthermore, MYC is known to possess repression activity on certain targets (23) , and the effects of MAD, if any, on such genes have not been evaluated. Resolution of these issues awaits the identification of MYC and MAD target genes involved in the control of the G1 cyclin/CDK complexes. Nonetheless, the BAP-Mad1 transgenic mice provide a system in which the effects of perturbing the Myc/Max/Mad network can be studied and provide a tool that is likely to be useful in understanding how this network influences the cell cycle and differentiation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Construction of the Mad1 Transgenes and Production of Transgenic Mice.
The murine Mad1 cDNA (54) was amplified by the pfu DNA polymerase (Stratagene) according to the manufacturer recommendation using the forward primer Mad115 5'-GAAGATCTGCAGGATGGCGACAGCCG-3' and the reverse primer Mad113 5'-GAAGATCTGAGGTGGAGAGGACTCTC- 3' encompassing the initiation and termination codon of Mad1, respectively. The 708-bp amplification product was digested by Bgl2 and cloned into the BamHI site of the human BAP transgenic vector pBAP (a gift from William L. Perry III and Nancy Jenkins, NIH, Bethesda, MD; Ref. 1000 units/ml; recombinant murine GM-CSF and rmIL-3 (Immunex), 1000 units/ml; 56 ). The orientation and the sequence of the Mad1 PCR product were verified by DNA sequencing.

ClaI digestion of pBAP-Mad1 gave rise to a 5-kb fragment containing the Mad1 cDNA under the control of the human BAP. This fragment was injected into the pronuclei of F2 (C57BL6SJL/F1 x C57BL6SJL/F1) mouse embryos. Injected embryos were transferred into pseudopregnant females and allowed to develop to term.

Mice were screened at 1–2 weeks of age by PCR on genomic DNA extracted from toes or tails (84) . Genotyping of embryos was performed on DNA extracted from yolk sacs. PCR analysis was carried out using the following set of primers: MAD1H1 5'-AGAAGCTAAAGGGATTGGTACCG-3' and Mad113 5'-GAAGATCTGAGGTGGAGAGGACTCTC-3'. Only two lines transgenic for of pBAP-Mad1 could be obtained (B2 and B3). The mice used to generate the data presented in this study are F1 to F3 back-crosses into the C57BL/6J genetic background. Statistical analysis of weight was performed using a two-sided unpaired Student’s t test.

In situ hybridization experiments were performed according to Quéva et al. (30) .

Hematopoietic Cell Culture.
Hematopoietic cell culture was performed according to Foley et al. (54) . Cytokines from the indicated sources were used at the following concentrations: recombinant human granulocyte-colony stimulating factor (Amgen), 1000 units/ml; recombinant murine GM-CSF and rmIL-3 (Immunex), 1000 units/ml; baculovirus macrophage-colony stimulating factor (kind gift of Larry Rohrschneider), 1000 units/ml; and Epo (Amgen), 2 units/ml. Statistical analysis was performed using a two-sided unpaired Student’s t test.

Culture of Primary MEFs.
Primary MEFs were obtained using an established procedure (84) from embryos 13.5-p.c. days after conception that were either wild type or BAP-Mad1 transgenic (genotyping was performed on DNA isolated from a yolk-sac). In a culture plate 6 cm in diameter, 107 cells were initially seeded; the culture was designated as P1. They were then passaged rigorously at a density of 106 cells/6-cm plate every 3 days. P3 to P5 cells were used to collect the data. Cells were cultured at 37°C (5% CO2) in DMEM containing 10% FBS (Hyclone) supplemented with penicillin and streptomycin (Life Technologies, Inc.). For growth curves, dishes of 6 cm were seeded with 2.105 cells and counted after the indicated days with an hemocytometer; the media was changed every 2 days. For G0 synchronization by serum starvation, asynchronous cultures of 2.105 cells in a 6-cm plate (<50% confluence) were washed twice with PBS and placed in DMEM containing 0.1% FBS for 30 h. They were released from G0 block by replacing the media with DMEM, 10% FBS and analyzed by FACS. The cells were stained with propidium iodine as described (52) and analyzed on a FACScan II (Becton Dickinson) followed by data analysis with the MultiCycle or ModFit software. A significant proportion of tetraploid cells were encountered even in the early passage of our MEF culture. Therefore, we only took into account the G1 and S fraction of the diploid population and disregarded the analysis of the G2 fraction contaminated by tetraploid cells in G1.

To infect MEFs with LXSN retroviruses encoding 16E7 proteins, early passage cells were plated out at a density of approximately 3 x 105 cells in a 60-mm tissue culture dish. Twenty-four h later, cells were incubated with retroviral supernatants from PA317 cells at a titer of 2.4 x 106 infectious particles/ml in media containing Polybrene at a final concentration of 8 µg/ml. Forty-eight h after infection, G418 was added to a final concentration of 500 µg/ml (effective) and maintained at this concentration for the remainder of the experiment. Ninety-six h after infection, cells were replated at a density of 5 x 104 cells/35-mm tissue culture dish, and flow cytometry and cell counts were performed 72 h later.

Primary Antibodies.
All antibodies were obtained from Santa Cruz biotechnology except anticyclin E1, anticyclin A, anti-p27Kip1 (kind gifts of J. M. Roberts), anticyclin D1, D2, and D3 (kind gifts of C. Scherr), Mab 1, and anti-Rb (Zymed).

Western Blotting Analysis.
Exponentially growing cells at 50% confluence were lysed on the plate in 20 mM Tris (pH 7.4), 50 mM NaCl, 0.5% NP-40, 0.5% deoxycholate, 0.5% SDS, 1 mM EDTA, containing protease-inhibitors, followed by scraping and sonication. Protein concentrations were determined by Bradford assays, and 80 µg of cell extracts were resolved by SDS-PAGE and transferred on polyvinylidene difluoride membranes (MSI) as described previously (85) . Membranes were probed with the indicated antisera in TNT 0.5 buffer [0.15 M NaCl, 25 mM Tris (pH 7.5), 0.5% Tween 20] containing 1% nonfat dry milk. The membranes were washed four times with TNT 0.05 buffer (0.05% Tween 20) and incubated with horseradish peroxidase-conjugated donkey antirabbit (Amersham) or rabbit antimouse (Zymed) secondary antibodies. The membranes were washed four times in TNT 0.05 buffer, and analyzed by enhanced chemiluminescence (Amersham) procedures.

In Vivo Immunoprecipitation Kinase Assay.
Exponentially growing cells at 50% confluence were lysed on the plate in 25 mM Tris (pH 7.4), 125 mM NaCl, 2.5 mM EDTA, 0.05% SDS, 0.5% NP40, 0.5% deoxycholate, 10% glycerol, protease, and phosphatase inhibitors. The lysates were cleared by centrifugation and frozen. Cyclin E1, cyclin A, and CDK2 were immunoprecipitated from lysates containing 200 µg of proteins. The immunoprecipitates were washed three times with the lysis buffer and once with the kinase buffer [20 mM Tris (pH 7.4), 7.5 mM MgCl2, 1 mM DTT]. After the last wash, they were resuspended in 30 ml kinase buffer containing 4 mg histone H1, 50 mM ATP, and 5µCi {gamma}32P ATP and incubated for 30 min at 37°C (85) . pRb kinase to determine the activity of cyclin D1/CDK4 complexes was conducted similarly, except that the cells were lysed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween 20, 10% glycerol, 1 mM DTT, and protease and phosphatase inhibitors, and the kinase reaction was executed for 30 min at 30°C in 50 mM Hepes (pH 7), 10 mM MgCl2, 2.5 mM EGTA, 1 mM DTT, containing 2 µg of glutathione S-transferase-pRb, 50 µM ATP, and 5 µCi {gamma}32P ATP (86) .


    Acknowledgments
 
We are deeply indebted to Dr. D’Ercole (University of North Carolina at Chapel Hill) and Professor Frohman (University of Illinois at Chicago) for the measurements of the serological levels of insulin-like growth factors and growth hormone, respectively, to Keesook Lee for expert work in the generation of the transgenic mice, and to Carla Grandori and Nancy Jenkins for reagents and advice. We thank Bruce Clurman, Brian Iritani, and Lenora Loo for critical readings of the manuscript. We are also grateful to Santa Cruz Biochemicals for help with antibodies, to Dr. Barbara Johnston and the technicians of the Fred Hutchinson Cancer Research Center animal facility, Teresa Barton, Celine Lawler, Rebecca Walsh, and Gretchen Poortinga for assistance with animal care and photography at Peter MacCallum Cancer Institute, and to Dr. George Sale and the Fred Hutchinson Cancer Research Center pathology service.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Damon Runyon-Walter Winchell Foundation Postdoctoral Fellowship (DRG076) and a Special Fellowship of the Leukemia Society of America (to G. A. M.); INSERM and the Phillippe Foundation (to C. Q.), NIH/National Cancer Institute RO1 57138; and P50HL54881 (to R. N. E.). R. N. E. is an American Cancer Society Research Professor. Back

2 Present address: AstraZeneca Transgenic Center, AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden. E-mail: christophe.queva{at}astrazeneca.com Back

3 Present address: Peter MacCallum Cancer Institute, Division of Hematology and Medical Oncology, Locked Bag 1, A’Beckett Street, Victoria, Australia 8006. E-mail: g.mcarthur{at}pmci.unimelb.edu.au Back

4 To whom requests for reprints should be addressed, at Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Mailstop A2-025, P. O. Box 19024, Seattle, WA 98109-1024. Phone: (206) 667-4445; Fax: (206) 667-6522; E-mail: eisenman{at}fred.fhcrc.org Back

5 The abbreviations used are: CDKI, cyclin-dependent kinase inhibitor; pRb, retinoblastoma protein; BAP, ß-actin promoter; BrdUrd, bromo-deoxyuridine; BFU-E, burst-forming unit, erythroid; CFU-E, colony-forming unit, erythroid; CFU-G, colony-forming unit, granulocytic; CFU-GM, colony-forming unit, granulocytic-monocytic; CFU-M, colony-forming unit, monocytic; FACS, fluorescence-assisted cell sorting; GM-CSF, granulocyte macrophage colony-stimulating factor; GH, growth hormone; HPV, human papilloma virus; IGF-I, insulin-like growth factor 1; MEF, murine embryonic fibroblast; Rb, retinoblastoma; RT-PCR, reverse transcription-PCR; FBS, fetal bovine serum; Epo, erythropoietin. Back

6 C. Quéva, unpublished observation. Back

7 D. Galloway, Fred Hutchinson Cancer Research Center, personal communication. Back

8 L. James, R. N. Eisenman, unpublished result. Back

Received for publication 7/22/99. Revision received 8/24/99. Accepted for publication 9/27/99.


    References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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