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Cell Growth & Differentiation Vol. 10, 73-86, February 1999
© 1999 American Association for Cancer Research

Malignant Transformation of p53-deficient Astrocytes Is Modulated by Environmental Cues in Vitro1

Oliver Bögler2, Motoo Nagane, Jennifer Gillis, H-J. Su Huang and Webster K. Cavenee

Ludwig Institute for Cancer Research, La Jolla, California [O. B., M. N., H-J. S. H., W. K. C.], and Department of Anatomy and Division of Neurosurgery, Virginia Commonwealth University, Richmond, Virginia 23298 [O. B., J. G.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The early incidence of p53 mutation in astrocytomas suggests that it plays an important role in astrocyte transformation. Astrocytes isolated from homozygous p53 knockout mice grow rapidly, lack contact inhibition, and are immortal. Here we tested whether the loss of p53 is sufficient for progression to tumorigenicity of astrocytes. We grew primary astrocytes under three conditions for over 120 population doublings and assessed their antigenic phenotype, chromosome number, and expression of glioma-associated genes as well as their ability to form colonies in soft agarose and tumors s.c. and intracranially in nude mice. Under two conditions (10% FCS and 0.5% FCS plus 20 ng/ml EGF), cells acquired the ability to form colonies in soft agarose and tumors in nude mice, and this was accompanied by the expression of genes, including epidermal growth factor receptor, platelet-derived growth factor receptor {alpha} and ß, protein kinase C{delta}, and vascular endothelial growth factor, which are known to be aberrantly regulated in human astrocytomas. Under the third condition (0.5% FCS plus 10 ng/ml basic fibroblast growth factor), astrocytes gained the ability to form colonies in soft agarose and had abnormal chromosome numbers similar to cells in the first two conditions but did not form tumors in nude mice or overexpress glioma-associated genes. These data provide experimental evidence for the idea that the malignant progression initiated by the loss of p53 may be subject to modulation by extracellular environmental influences.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The loss of the tumor suppressor gene p53 is the earliest and most common genetic lesion in astrocytic gliomas described to date (1) . Some 35–40% of astrocytomas have p53 mutations (2) , and many of those that have wild-type p53 carry mdm2 amplifications, which functionally inactivate p53 (3) . Because the frequency with which p53 mutations are found in astrocytic gliomas does not significantly increase with higher grades, it has been suggested that p53 mutations occur early in the progression of these tumors. Moreover, evidence is accumulating that p53-deficient astrocytomas may represent those with apparent evolutionary stages that take place over an extended period of time (4, 5, 6) .

The high frequency and early occurrence of loss of wild-type p53 function prompted the examination of the impact of the loss of p53 on the behavior of primary astrocytes, a presumptive precursor cell for astrocytic gliomas. Freshly isolated astrocytes from homozygous p53 knockout mice showed more rapid growth than astrocytes from wild-type or heterozygous littermates and a striking absence of contact-mediated growth inhibition (2 , 7) . After continuing growth in culture, p53-/- astrocytes acquired the ability to form colonies in soft agarose (2) and tumors in nude mice (7) .

These results suggested that the loss of the p53 tumor suppressor gene could initiate in vitro progression and that in the presence of a continuous growth stimulus, could be sufficient to drive astrocytes to a tumorigenic phenotype. Continuous cell division is strongly associated with the malignant state, and the presence of a constant growth-promoting stimulus may be an important prerequisite for malignant progression. Highly malignant glioma cells that can be shown to be independent of exogenous growth factors are no exception, as they usually engage in autocrine growth factor production (8) , or have acquired a mutated and constitutively active growth factor receptor (9) . However, it is less well understood whether the presence of any growth-promoting signal is sufficient to allow progression to malignancy, or whether only some of the growth factors which are able to cause division in a given cell type are able to promote this progression.

The general importance of growth factors, their receptors, and signaling machinery in cancer is abundantly evident, from the fact that many are encoded by proto-oncogenes (10) , and the ability of some growth factors to cause transformation-like states in cultured cells, including glial cells (11) . Studies on the role of growth factor signaling in development have demonstrated that different growth factors can have profoundly different influences on the division and differentiation choices of normal cells. For glial cells, this is best illustrated by the different effects that PDGF,3 bFGF, and a combination of both have on the propensity of oligodendrocyte type-2 astrocyte progenitor cells to undergo self-renewal or differentiation (12, 13, 14, 15) . This raises the possibility that malignant progression may be the consequence of differing growth factor concentrations or combinations present in the extracellular milieu at the time a cancer precursor cell undergoes an initiating mutation.

Here we sought to directly test this idea by determining the rates of malignant progression in astrocytes that had the same initiating mutation, the loss of p53, but which were grown under different culture conditions. Primary astrocytes were maintained under different growth conditions, each of which has been previously shown to support the proliferation of astrocytes (16 , 17) . The growth rate, in vitro transformation, and ability of the cells to form tumors were determined at various times over 120 population doublings. Although cells in all three conditions were aneuploid, immortal, and acquired the ability to form colonies in soft agarose, there was a differential effect on progression to the ability to form tumors. The inappropriate expression of several genes implicated in human gliomas, including the EGFR, PDGFRs {alpha} and ß, and PKC{delta}, correlated with tumorigenic capacity in these cells. These data suggest that stimulation of cell division in the presence of a predisposing genomic instability is not sufficient to guarantee malignant progression, and that the nature of the growth-promoting stimulus may play a significant role.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Growth Characteristics and Antigenic Phenotype of p53-/--Astrocytes.
p53-/- astrocytes were purified from neonatal mouse cortices and either grown in DMEM containing 10% FCS (DMEM-FCS), DMEM containing 0.5% FCS supplemented with 20 ng/ml EGF (DMEM-EGF), or DMEM containing 0.5% FCS supplemented with 10 ng/ml bFGF (DMEM-bFGF). Cells were plated and passaged according to a standard schedule; 100,000 cells were plated per 10-cm dish, in triplicate, and grown for 6–8 days before being passaged and replated (see "Materials and Methods"). This regimen allowed cultures to reach high density while maintaining exponential growth. When population doublings were determined at each passage, counted cumulatively, and plotted against time (Fig. 1)Citation , exponential growth was maintained in all three conditions and did not show kinetics, indicating entry into senescence. Growth rates in DMEM-FCS were consistently higher than in DMEM-EGF or DMEM-bFGF, and cells in DMEM-bFGF grew slightly slower than cells in DMEM-EGF after the first 50 population doublings. In DMEM-FCS, cells achieved 100 population doublings in {approx}125 days, or an average cell cycle time of 30 h.



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Fig. 1. Growth rate of p53-/- astrocytes. A graph of time against calculated cumulative population doublings shows that the p53-/- astrocytes in DMEM-FCS, DMEM-EGF, and DMEM-bFGF grew exponentially throughout their time in culture and showed no senescent or critical phase. The growth rate in all three conditions was similar over time, although cells in DMEM-FCS grew consistently faster. Population doublings were calculated at every passage from the numbers of cells obtained in triplicate platings of 100,000 cells.

 
p53-/- astrocytes were immunoreactive for GFAP, showing strong filamentous staining initially, and lower levels of diffuse staining after about five passages (Ref. 18 and data not shown). Some of them were also labeled by the monoclonal antibody A2B5 (19) , which has been shown previously to recognize a subset of astrocytes in the developing spinal cord (20) as well as multipotent glial progenitor cells (21) . To characterize the effect of growth conditions on antigenic phenotype, cells at every other passage from P3 to P29 were stained with A2B5, and the proportion of positive cells was counted (Fig. 2)Citation . At passage 0, immediately following purification and before cells were switched into either DMEM-bFGF or DMEM-EGF, <5% of cells were A2B5+. However, when p53-/- astrocytes were cultured in DMEM-bFGF, the proportion of A2B5+ cells rapidly increased, going through a period of fluctuation before resolving to >95% A2B5+ (Fig. 2)Citation . In contrast, few cells grown in either DMEM-FCS or DMEM-EGF were A2B5+ (Fig. 2)Citation . To test whether the A2B5+ nature of astrocytes in DMEM-bFGF could be altered by environmental conditions, cells at passage 29 were switched into DMEM-FCS. Within 2 weeks of the switch, <5% of the cells were A2B5+, suggesting that this antigenic characteristic is dependent on environmental signals.



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Fig. 2. The percentage of p53-/- astrocytes that are A2B5 positive in different growth conditions. At each passage, cells were plated in triplicate on coverslips and stained with the monoclonal antibody A2B5. Cultures in DMEM-FCS (A) and DMEM-EGF (B) showed a consistently small number of cells that were A2B5 positive, whereas in DMEM-bFGF (C) the percentage grew to >90%. The percentage of positive cells was obtained by counting over 200 cells/coverslip. Data are means; bars, SD.

 
Aneuploidy Develops Rapidly in p53-/- Astrocytes.
The lack of p53 is associated with loss of cell cycle checkpoints, resulting in the instability of the genome, including abnormalities in chromosome number (22 , 23) . Metaphase spreads were prepared from astrocytes grown in all three conditions at early and late passages, and chromosomes were counted (Fig. 3)Citation . Even at passage 5, most cells in any of the three conditions showed abnormal ploidy. For example, in a population of 92 metaphases from cells cultured in DMEM-FCS for five passages (Fig. 3A)Citation , only 3 had close to the normal diploid number of 40 chromosomes. The remaining 89 cells had more chromosomes and were distributed in two peaks, one below 80 and one below 160, or 4n and 8n, respectively. p53-/- astrocytes grown in DMEM-FCS divided most rapidly and therefore provided the largest number of countable metaphases. However, the lower number of countable metaphase spreads obtained from cells grown in DMEM-EGF and DMEM-bFGF at passage 5 also suggested abnormal ploidy. At late passages, significant numbers of metaphases were obtained in all growth conditions and showed abnormal numbers of chromosomes, with the majority of cells having {approx}80 chromosomes or close to 4n (Fig. 3, B, D, and F)Citation .



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Fig. 3. p53-/- astrocytes have abnormal numbers of chromosomes. G-banded chromosomes were prepared from cells in DMEM-FCS (A and B), DMEM-EGF (C and D), and DMEM-bFGF (E and F) after 5 (A, C, and E) or 29 (B, D, and F) passages. All countable metaphase spreads were included in the analysis. Chromosomes were counted under the microscope and charted on the histograms. The total number of metaphase spreads obtained in each group is indicated. Few cells possessed the expected 40 chromosomes.

 
p53-/- Astrocytes Become Transformed with Time in Culture.
To test whether the lack of p53 leads astrocytes to accumulate phenotypic changes associated with malignancy, their ability to form anchorage-independent colonies in soft agarose was determined. p53-/- astrocytes, cultured in the three conditions described above, were taken at every other passage, placed in soft agarose, and cultured for 2 weeks; then the number of colonies was counted. To compensate for the slight differences in growth rate between p53-/- astrocytes grown in DMEM-FCS, DMEM-EGF, and DMEM-bFGF (see Fig. 1Citation ), the data in Fig. 4Citation are plotted against cumulative population doublings. Cells that were grown in DMEM-FCS or DMEM-bFGF acquired the ability to form colonies in soft agarose after about 50 population doublings, with the yield being consistently higher for cells grown in DMEM-FCS than those grown in DMEM-bFGF. DMEM-EGF-cultured p53-/- astrocytes did not form similar numbers of colonies in soft agarose until they had undergone close to 100 population doublings, and high levels of colonies were not obtained until after 150 population doublings. Thus, the growth conditions of astrocytes greatly influenced their abilities to acquire the ability to grow in an anchorage-independent manner.



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Fig. 4. Colony formation in soft agarose by p53-/- astrocytes. The efficiency of colony formation in soft agarose per 1000 cells is shown against cumulative population doublings. Cells were plated in soft agarose at every other passage and allowed to grow for 2 weeks before the numbers of colonies were scored. The passage number was converted to cumulative population doublings using the graph in Fig. 1Citation . Data are means of triplicate platings of three different cell concentrations; bars, SE. A, DMEM-FCS; B, DMEM-EGF; C, DMEM-bFGF.

 
s.c. Tumor Formation in Nude Mice.
An important measure of in vitro progression toward malignancy by p53-/- astrocytes is their ability to form tumors in experimental animals. To exclude the influence of the immune system, nude mice were used as hosts. Initially, p53-/- astrocytes were injected into the flanks of nude mice, a site in which tumor growth is easier to monitor while the animal is alive. Furthermore, because larger numbers of cells can be injected s.c. than at other sites, these experiments afford the best opportunity for cells with low efficiencies of tumor formation to do so. One million cells were injected into each flank, and the appearance and growth of tumors were monitored by palpation and measurement.

p53-/- astrocytes grown in DMEM-FCS formed s.c. tumors at passage 5 (about 15 population doublings), the earliest tested, at a frequency of {approx}30% (Table 1)Citation . At passage 9 (or 45 population doublings) and higher, DMEM-FCS-grown astrocytes were s.c. tumorigenic at a frequency of 100%. Although p53-/- astrocytes grown in DMEM-EGF also acquired the ability to form tumors s.c., they did so only after >15 passages (65 population doublings; Table 1Citation ). In stark contrast to the behavior of cells in these two conditions, p53-/- astrocytes grown in DMEM-bFGF never formed tumors at any passage tested, including passage 29 (>120 population doublings; Table 1Citation ), when colony formation in soft agarose was maximal (Fig. 4)Citation . Mice receiving injections of cells grown in DMEM-bFGF were observed for >120 days before sacrifice and direct confirmation of the absence of tumor formation.


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Table 1 s.c. tumor formation in nude mice

 
The latency and rate with which s.c. tumors grew was also a factor of passage number. DMEM-FCS grown cells at passages 15/17 or 29 showed tumor growth rates that were similar to each other and faster than passage 9 cultures of the same cells (Fig. 5A)Citation . DMEM-FCS grown astrocytes at passage 5 showed even slower growth and longer latencies and, in addition, were quite variable in the behavior of individual tumors. These are shown individually (Fig. 5ACitation , dotted lines). Interestingly, the latency of s.c. tumors derived from cells grown in DMEM-EGF was noticeably longer than for DMEM-FCS passage 9 or higher cells (Fig. 5B)Citation , although these cells also formed s.c. tumors with 100% efficiency (Table 1)Citation . The observation that passages 21 and 29 were similar in tumor growth rate to those derived from DMEM-FCS astrocytes once they appeared suggests that they required longer to overcome a growth-limiting constraint, rather than necessarily having a slower growth rate per se. Even the most rapidly growing s.c. tumors derived from p53-/- astrocytes formed tumors more slowly than the human glioma cells line U87MG (Fig. 5)Citation , suggesting that the limited growth rate and latency displayed by these cells were an inherent factor rather than representing a limit imposed by the mouse-flank environment.



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Fig. 5. s.c. tumor formation in nude mice by p53-/- astrocytes. The size of tumors resulting from injections of 1 x 106 cells s.c. was measured regularly, and the calculated volume is charted against days after injection. Tumors derived from p53-/- astrocytes in DMEM-FCS (A) or DMEM-EGF (B) are shown along with the highly tumorigenic glioma cell line U87MG (B). The abrupt changes in volume between the last and next-to-last measurements are due to underestimates of the tumor volume before dissection caused by the invasion of the pleural cavity by tumors. Tumors from DMEM-FCS cultured cells after five passages are graphed individually (dotted lines) because of the high degree of variability that they showed in growth rate. Other tumor groups are graphed as means; bars, SD. Error bars are sometimes smaller than the plot symbols.

 
s.c. tumors derived from U87MG cells were encapsulated and soft to the touch and moved freely relative to the mouse skin and ribcage. In contrast, one-half of the DMEM-FCS passage 15/17-derived tumors and all DMEM-FCS passage 29-derived tumors became hard to the touch and rigidly attached to the ribcage over time. Postmortem dissection revealed that these tumors had invaded the pleural cavity and often caused compression of the lungs and heart. Consequently, their sizes had been somewhat underestimated at later stages of growth during measurements of the live animal, accounting for the steep gradient between the last two data points of curves for tumors derived from cells grown in DMEM-FCS for 9 or more passages (Fig. 5)Citation . This apparently aggressive, invasive behavior, therefore, also appeared to be a function of time in culture.

Intracranial Tumor Formation in Nude Mice.
A more relevant environment in which to test the ability of malignant astrocytes to form tumors is the brain. Therefore, 5 x 105 p53-/- astrocytes grown in different conditions for various times were injected intracranially. The efficiency of intracranial tumor formation was similar to that seen previously in the flank (Table 2)Citation . Low-passage DMEM-FCS grown cells showed noticeably lower efficiencies than higher passage cells, which came close to 100%. DMEM-EGF cells at passage 15 and above formed tumors with a 100% efficiency, revealing the only difference between intracranial and s.c. injections, where passage 15 cells had failed to form a tumor. Astrocytes grown in DMEM-bFGF failed to form intracranial tumors, even at passage 29.


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Table 2 Intracranial tumor formation in nude mice

 
In some animals, the first indications of a tumor were weight loss, inactivity, and spinal curvature. In other animals, an extracranial and s.c. tumor formed, or the eye ipsilateral to the injection site was enucleated, forcing the sacrifice of the animal. These extracranial tumors formed, although the burr-hole in the skull was sealed with bone wax after injection, and may be related to the highly invasive nature of p53-/- astrocytes as described above for s.c. injections. The time at which tumor-related symptoms first appeared varied between 29 days postinjection for a tumor derived from DMEM-FCS passage 29 cells to 173 days postinjection for a tumor obtained from DMEM-FCS passage 5 cells. Tumor formation was confirmed in all cases by sectioning of the brain and histological staining.

Prior to intracranial injection, cells were genetically labeled with the gene for the human PLAP enzyme using the {psi}2-DAP retrovirus (24) to facilitate their identification after injection (Fig. 6, A and B)Citation . Cultures were >99% positive for PLAP before injections (data not shown). Histological examination of frozen sections of tumor-bearing mouse brain, following reaction with the chromogenic substrate for PLAP and counterstaining with H&E, greatly facilitated the identification of tumor cells, even when they had left the predominant tumor mass. Examination of PLAP/H&E-stained tumor-bearing brain sections revealed that the brain tumors formed by p53-/- astrocytes displayed some of the characteristic histological features of malignant human gliomas (Fig. 6)Citation . p53-/- astrocytes formed tumors that were invasive and showed no encapsulation, although the overall compactness of the tumors varied. In some cases, the tumor mass was largely found near the injection site, as in two of the tumors shown: the tumor derived from cells grown in DMEM-EGF for 29 passages (Fig. 6B)Citation or DMEM-FCS for 29 passages (Fig. 6I)Citation . In all such cases, the tumor margins were ragged due to the infiltration of the normal brain parenchyma by peripheral cells, which were found short distances from the tumor mass, surrounded on all sides by normal brain (Fig. 6, C and D)Citation . In some cases, infiltration resulted in the tumor pervading an entire hemisphere at a given plane of section. A good example of this is the one brain tumor obtained from DMEM-FCS astrocytes at passage 5. The injection site is clearly visible under low magnification (Fig. 6E)Citation , as the apparently necrotic region toward the upper right hand side of the panel, and can be seen to lie within the area of highest tumor cell density. PLAP-positive cells can be seen throughout the hemisphere and, under higher magnification (F and G), appear as groups of cells surrounded by normal brain parenchyma. It appears that some PLAP-positive tumor cells are beginning to invade the contralateral hemisphere (Fig. 6G)Citation . Overall, the density of tumor cells appears to diminish as a factor of distance from the injection site. In the least compact tumors, near-uniform infiltration of the normal brain by PLAP-positive cells was observed, accompanied by smaller regions of high tumor cell density. For example, the tumor derived from astrocytes grown in DMEM-FCS for 17 passages shown in Fig. 6HCitation had infiltrated the entire hemisphere shown and showed one area of tumor mass that was far away from the presumed injection site.



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Fig. 6. p53-/- astrocytes form invasive and vascularized intracranial tumors in nude mice. Frozen sections of brain tumors formed after injection of 5 x 105 p53-/- astrocytes were stained with H&E only (A) or for alkaline phosphatase and counterstained with H&E (B–K). Cells were transduced with a retrovirus encoding the human PLAP gene before injection. A and B, nearby sections of tumor derived from cells grown in DMEM-EGF for 29 passages stained with H&E alone (A) or for alkaline phosphatase as well (B) showing the ability of alkaline phosphatase to reveal the area of the tumor. Note that the skull fragments, which remained firmly attached to the tumor, stain dark purple with hematoxylin alone in A. C and D, a higher power image of the margin of another tumor derived from DMEM-EGF grown cells after 29 passages, showing that the tumor margin is ragged, suggesting cells are invading the normal brain parenchyma. E–G, successive magnifications of a tumor derived from DMEM-FCS cells grown for five passages, showing the area where the tumor crossed to the contralateral hemisphere. Note the area of apparent necrosis around the injection site in E. H, a tumor derived from DMEM-FCS astrocytes after 17 passages, showing the high degree of invasion revealed by the alkaline phosphatase positive cells seen throughout the hemisphere. I and J, a highly angiogenic tumor derived from DMEM-FCS cells after 29 passages showing evidence of hemorrhage and invasion around the periphery. K, colocalization of alkaline phosphatase-positive cells with a blood vessel in a DMEM-FCS passage 17-derived tumor. Note also the vessel seen in cross section, which also has alkaline phosphatase-positive cells associated with it. Bar: A–C and E–J, 1 mm; D and K, 0.5 mm.

 
Tumors derived from p53-/- astrocytes were often associated with areas of apparent hemorrhage, as shown in Fig. 6, I and JCitation . This was particularly common in tumors derived from passage 29 DMEM-FCS-grown cells, and often hemorrhage was found at the periphery of the tumor mass. In addition, a close apposition of PLAP-positive tumor cells and blood vessels could be observed in several tumors (Fig. 6K)Citation , allowing for the possibility that tumor cells were inducing neovascularization or migrating along blood vessels.

Alterations in the Expression of Genes Implicated in Human Glioma Correlate with the Ability to Form Tumors.
As a first step toward investigating the molecular underpinnings of the differences in tumorigenic capacity of p53-/- astrocytes and the similarity of the process to gliomagenesis in humans, we studied the expression of genes implicated previously in the progression of human gliomas. Total cellular RNA isolated from cells at every other passage, in each growth condition, was subjected to Northern blotting. In addition, RNA from early-passage astrocytes of all three p53-knockout genotypes as well as from s.c. tumors derived from DMEM-FCS and DMEM-EGF grown cells were analyzed (Fig. 7Citation and Table 3Citation ). RNA loading was controlled for by probing for GAPDH mRNA. The signal intensities were quantified by densitometry and normalized to the GAPDH signal.



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Fig. 7. Northern analysis of gene expression in p53-/- astrocytes. Autoradiographs of Northern analysis of total cellular RNA from p53-/- astrocytes in the growth conditions and at the times indicated and from s.c. tumors derived from these cells. For details of the probes used, see "Materials and Methods."

 

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Table 3 Relative level of gene expression in p53-/- astrocytes and s.c. tumors derived from them

 
Several genes showed differential expression between p53-/- astrocytes in different conditions or at different times in culture. For example, the mRNAs for the EGFR (25) , PDGFR-ß (26) , PDGFR-{alpha} (27) PKC-{delta} (28) , and VEGF (29) were detectable only in cells grown in DMEM-FCS or DMEM-EGF and not in cells grown in DMEM-bFGF (Fig. 7Citation Table 3Citation ). The mRNA for PDGFR-ß was detectable in p53+/+, p53+/-, and p53-/- astrocytes at passage 1, suggesting that it is the maintenance of expression in DMEM-FCS and the reexpression in DMEM-EGF that set these cultures apart from the lack of expression seen in DMEM-bFGF-grown cells. Overexpression of the EGFR and PDGFR-{alpha} and -ß genes has been described in human gliomas, both in the presence and in the absence of genomic amplification (30 , 31) . No genomic amplification (beyond the increase in ploidy described above) or deletion was detected in p53-/- astrocytes at any passage or in any condition by Southern blot analysis for any of the genes discussed here (data not shown). The expression of these genes therefore correlated with the ability of p53-/- astrocytes to form tumors.

The tumors generated by p53-/- astrocytes were highly vascularized (Fig. 6)Citation , suggesting that they had the ability to induce the formation of new blood vessels. In agreement with this, astrocytes cultured in DMEM-FCS and DMEM-EGF expressed mRNA for the angiogenic factor VEGF, and the mRNA for bFGF (32) was observed in tumors derived from p53-/- astrocytes, although not in cultured cells.

Two other differences in gene expression between different cultures of p53-/- astrocytes were observed but did not correlate with tumorigenicity. The met-1 gene (33 , 34) was expressed by all astrocytes at low levels and became relatively overexpressed in p53-/- astrocytes in all culture conditions with time. This is similar to what has been observed previously in fibroblasts derived from p53-/- mice (35) . However, in astrocytes this overexpression did not correlate with the ability to form tumors. Lastly, the gene for the EGFR ligand, TGF-{alpha} (34) , was also detectable in all p53-/- astrocytes in culture after passage 1. Interestingly, this gene became relatively overexpressed in late-passage cells grown in DMEM-bFGF, the one condition in which cells did not receive exogenous EGFR ligand beyond what is present in 0.5% FCS. The overexpression of this gene therefore showed an inverse correlation with the ability of cells to form tumors, in contrast to its association with increased malignancy in human tumors (36) .

The expression of other genes analyzed was detected but did not vary between cells in different conditions or at different times. These included the retinoblastoma gene (37) , p15INK4b (38) , p16INK4a (38) , Cdk4 (39) , bax (40) , Fgfr1 (41) , gli (42) , Dep-1 (43) , and src (44) . Lastly, no mRNA for the following genes was detectable in any of the astrocytes by Northern blotting: p21 (45) , PDGF-B (46) , PKC-{zeta}, PKC-{theta}, and PKC-{eta}(47 , 48) .


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The loss of wild-type p53 activity, either by direct mutation or by amplification of the mdm-2 gene (3) , is the most common and earliest known genetic lesion in astrocytic gliomas (2) . The implication that this loss plays a central and early role in the initiation and progression of astrocytomas was tested by examining astrocytes from p53-/- mice in culture. After purification (49) , a clonally complex population of pure cortical astrocytes was split into three different culture conditions, DMEM-FCS, DMEM-EGF, or DMEM-bFGF, and maintained in serial passage for over 120 population doublings. In all three conditions, p53-/- astrocytes exhibited exponential growth, in the absence of crisis and without a slowing of growth rate that would indicate the onset of cellular senescence. This suggests that the loss of p53 is either sufficient for the immortalization of astrocytes or rapidly leads to the accumulation of secondary events responsible for the removal of limitations on astrocyte division, as has been suggested for fibroblasts (50) .

To examine whether the loss of wild-type p53 activity also led astrocytes to acquire the ability to form tumors, cells were injected into nude mice after various times in culture. Although the growth rate and the rate of acquisition of the ability to form colonies in soft agarose was similar in all three conditions, cells differed markedly in their ability to form tumors. p53-/- astrocytes grown in DMEM-FCS and DMEM-EGF progressed to a tumorigenic phenotype after a different number of population doublings. In DMEM-FCS, it took only 15 population doublings to reach 30% efficiency in s.c. tumor formation and 45 doublings to gain 100%. In DMEM-EGF, the earliest cells tested that formed tumors had been in culture for 50 population doublings. The most striking difference observed was that p53-/- astrocytes grown in DMEM-bFGF never formed tumors, even after 120 population doublings either s.c. or intracranially. These data demonstrate that the loss of p53 itself is not sufficient to confer tumorigenicity on astrocytes and suggest that its loss may also not inevitably lead to a fully malignant phenotype.

In addition to differing in their ability to form tumors, p53-/- astrocytes differed in the genes that they expressed. A survey of genes that have been implicated in the progression of human gliomas (51, 52, 53) revealed that measurable expression of the EGFR, PDGFRs, VEGF, and PKC-{delta} was observed only in cells grown in DMEM-FCS and DMEM-EGF. Therefore, the detectable expression of these glioma-associated genes correlated with the ability of p53-/- astrocytes to form tumors. The products of these genes are involved in the growth, survival, and angiogenesis-related signaling that is associated with the malignant behavior of gliomas, and therefore their up-regulation in p53-/- astrocytes cultured in DMEM-FCS or DMEM-EGF may contribute to the malignant phenotype of these cells. The absence of detectable expression of these genes in p53-/- astrocytes in DMEM-bFGF suggests that this up-regulation is not a simple and direct consequence of the loss of wild-type p53 activity but represents separate steps in the malignant progression of astrocytes. The similarity in tumor progression-associated gene expression changes between p53-/- astrocytes cultured in DMEM-FCS or DMEM-EGF and human astrocytomas demonstrates that significant aspects of the molecular progression of astrocytoma can occur in vitro. Furthermore, the controlled conditions in which p53-/- astrocytes can be grown suggest that they provide a useful model system in which to test the impact of the misexpression of these glioma-associated genes and their interrelationship with the loss of p53 function.

Overexpression in human gliomas of both PDGFR-{alpha} (54) and PDGFR-ß (55) occurs frequently and often in the same tumor (31) . This overexpression is usually found in the absence of genomic amplification and often follows p53 loss or mutation (56 , 57) . Detailed analysis of gliomas using in situ hybridization techniques has revealed that PDGFR-{alpha} appears to be expressed in the glial component of the tumor and PDGFR-ß in the endothelial cell component (58) . Our observation of PDGFR-ß mRNA in p53-/- astrocytes may represent a difference between cultured cells and cells in the brain or a species difference. The observation that passage 1 astrocytes of any p53 genotype express the PDGFR-ß suggests that this may be a characteristic of murine astrocytes.

A potential significant difference between the malignant progression of p53-/- astrocytes in culture and human astrocytic gliomas is the lack of abnormalities in the retinoblastoma pathway. Inactivation of the retinoblastoma or p16INK4a protein or amplification of the cdk4 gene collectively occurs in as many 95% of human gliomas (53) . No alterations in mRNA levels of these genes, nor dosage alterations at the genomic level, were observed in p53-/- astrocytes. Although these data do not exclude the possibility that point mutations are occurring in these genes, as has been shown for the p16INK4a gene in human gliomas (59) , they do demonstrate that the more commonly found deletions, silencing or amplification, are absent. Therefore, p53-/- astrocytes in vitro appear to accumulate only some of the molecular changes identified in human gliomas. This limitation may be a consequence of the culture environment or represent a species difference.

The observation that p53-/- astrocytes undergo varying degrees of malignant transformation in different growth conditions raises the question of how the cellular environment influences tumor progression. The simplest mechanism that can be proposed for the in vitro progression of p53-/- astrocytes grown in DMEM-FCS or DMEM-EGF is that the loss of wild-type p53 allows the accumulation of secondary genomic mutations, in the absence of cell cycle arrest and DNA repair, or apoptosis (60) . In the absence of obvious exogenous genomic insults, these mutations would likely be caused by replication errors, and therefore are most likely to occur stochastically and accumulate as a factor of the number of times a cell divides. The observation that the ploidy of p53-/- astrocytes was abnormal, regardless of the growth condition, confirms that these cells have unstable genomes and therefore have the potential to accumulate additional mutations. According to this line of reasoning, the number of times a cell has divided, and not the nature of the stimulus that caused the divisions, would determine the degree of progression. Although this model is sufficient to explain the behavior of cells grown in DMEM-FCS and DMEM-EGF when considered individually, it fails to explain the differences in the rate of progression of these cells, nor why cells in DMEM-bFGF failed to progress to tumorigenicity altogether. Although it could be argued that the additional population doublings that cells in DMEM-EGF required to become tumorigenic are due to inherent variability in the rate of progression, this is unlikely to explain the total lack of tumorigenicity of cells in DMEM-bFGF, and therefore other explanations must be considered. The inability of cells grown in DMEM-bFGF to form tumors, even after 120 population doublings, demonstrates that simply maintaining p53-/- astrocytes in a growth-permissive environment is not sufficient to guarantee their acquisition of a fully malignant phenotype. This implies that the nature of the growth stimulus experienced by a genetically vulnerable population of cells can influence the rate and extent of their malignant progression.

Growth conditions could influence progression by selective or instructive means. Selection, resulting in populations of cells that differ in their tumorigenic capacity, could occur on the basis of cell lineage or sub-type. The starting population for these experiments is purely astrocytic, as judged by their expression of GFAP. However, a great deal of heterogeneity between astrocytes has been reported, although not in regard to their ability to undergo malignant transformation (20 , 61) . Therefore, it is possible that growth in DMEM-bFGF selects a lineage or subtype of astrocytes that has an inherently lower capacity to form tumors. The observation that growth in DMEM-bFGF, but not in DMEM-FCS or DMEM-EGF, produces a population of astrocytes that are nearly all A2B5+ would appear to support this contention. However, the observation that changing the growth conditions from DMEM-bFGF to DMEM-FCS causes cells to become A2B5- suggests that it is not a stable heritable lineage attribute. Furthemore, in a study of spinal cord astrocytes, clones that contained A2B5+ cells invariably also contained A2B5- cells, suggesting that A2B5 reactivity is not a reliable marker of lineage or astrocyte subtype (20) . Therefore, A2B5 is not a useful lineage marker in this instance, and therefore does not allow this question to be resolved at present. Resolution of this question will require a more complete classification of astrocytes, which is still outstanding due to a lack of robust antigenic markers. An alternative basis of selection of astrocyte populations with different tumorigenic capacities by growth conditions would be the nature of the mutations that they accumulate. In this hypothesis, DMEM-bFGF would fail to select constellations of mutations that confer tumorigenicity. The genetic alterations that give cells a growth advantage in DMEM-bFGF may not be compatible with tumor formation in vivo.

The second possibility is that growth conditions are instructive, determining the molecular changes that p53-/- astrocytes undergo in culture. Although at present no mechanisms by which growth factors can directly cause specific DNA mutations have been proposed, it is well established that they are powerful regulators of gene expression. Exposure to DMEM-FCS or DMEM-EGF might directly cause expression of the glioma-associated genes mentioned above, whereas DMEM-bFGF does not,, and therefore explain differences in tumor formation. However, several findings suggest that this simple explanation is not sufficient. Cells in all conditions expressed TGF-{alpha} and therefore presumably have EGFR ligand available to them. Also, the expression of glioma-associated genes in DMEM-EGF did not occur immediately, as would be expected from a gene that was simply activated by the appearance of an external signal. Lastly, when cells are placed in vivo, all cells receive similar signals regardless of their in vitro history, and therefore the ability to form tumors must now be inherent in the cells. One prediction of an instructive mechanism would be that altering in vitro growth conditions for a short time would alter the tumorigenic capacity of cells, a hypothesis that is being tested experimentally at present.

Regardless of the mechanism by which the extracellular environment influences the rate of malignant progression, it is clear that the loss of wild-type p53 function does not inevitably result in the complete malignant transformation of astrocytes. Furthermore, it suggests that the interplay between intrinsic cellular genetic damage and extrinsic growth stimuli is likely to be of significance to the transformation process.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Culture of p53-/- Astrocytes.
Cultures of p53-/- astrocytes were generated as described previously (18) . Briefly, a pair of heterozygous TSG-p53 mice (62) , each bearing one wild-type and one disrupted p53 allele (designated + and -, respectively) were obtained from Genpharm (Mountain View, CA) and mated to generate a litter containing all three genotypes: p53+/+, p53+/-, and p53-/-. Cortical astrocyte cultures were prepared and purified as described (18 , 49 , 63) . After purification and genotyping, the initial passage number was designated as 0, and cultures of p53-/- astrocytes were established by plating cells in triplicate at a density of 100,000 cells per 10-cm Petri dish, in three conditions; they either remained in DMEM containing 10% FCS or were switched gradually and stepwise to DMEM containing 0.5% FCS, which was supplemented with either 10 ng/ml recombinant bovine bFGF or 20 ng/ml recombinant human EGF (growth factors from Boehringer-Mannheim). DMEM containing 0.5% FCS only, with no supplemental factors, was unable to sustain the growth of these cells. p53-/- astrocytes were subjected to sequential passage, 100,000 cells per 10-cm Petri dish being plated in triplicate every 6–8 days. The medium was changed, and growth factors were supplied every other day. Cell number was assessed by counting after each passage and used to calculate population doublings. At intervals, cells were harvested for analysis by a variety of methods. RNA was prepared from cultured cells or tumors using Trizol reagent (Life Technologies, Inc.), according to the manufacturer’s instructions, and Northern analysis was performed according to standard protocols.

Antigenic Phenotype.
Antigenic phenotype was assessed periodically by immunocytochemistry using anti-GFAP and A2B5 antibodies as described previously (18) . All of the astrocytes studied were found to be GFAP positive, although the intensity of GFAP staining declined with time in culture, and became more diffuse, although when stained within six h of passaging, the majority of cells reacted strongly with anti-GFAP antibodies and showed the classic filamentous staining profile.

Chromosome Number.
Chromosome numbers were obtained by counting G-banded metaphase spreads prepared by standard methods (64) .

Colony Formation in Soft Agarose.
Growth of cells in soft agarose was monitored every other passage using a standard method (65) , except that when astrocytes that had been grown in DMEM-bFGF or DMEM-EGF and were transferred into soft agarose (SeaPlaque, Rockland, ME), they received 20-fold less serum than called for by the standard protocol and continued to receive growth factors every other day (18) .

Tumor Formation.
For s.c. tumor formation, 1 x 106 cells were injected s.c. into both flanks in 100 µl of PBS using a 26-gauge needle. Animals were observed daily, and tumors were measured every 2–3 days after they became visible. Intracerebral stereotactic inoculations were performed as described previously (66) ; 5 x 105 cells were resuspended in 5 µl of PBS and inoculated into the right corpus striatum of the brain of 4–5-week-old female nude mice of BALB/c background using a Hamilton syringe. Brains were removed at various time points and immersion fixed in 4% paraformaldehyde, embedded in OCT (Miles, Elkhart, IN), frozen on dry ice, and stored at -20°C. Cryostat sections (10–15 µm) were collected and immersed in 4% paraformaldehyde, washed in PBS, and heat treated in PBS at 65°C for 20 min. Then sections were immersed in AP buffer [100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50 mM MgCl2] for 10 min and stained with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (SigmaFast tablets; Sigma Chemical Co.) for 60–90 min. After a rinse in water, dehydration through an ethanol series, and rehydration to water, sections were counterstained in Harris’ H&E.

Northern Blotting.
32P-labeled, random-primed DNA probes were prepared from inserts obtained from the following plasmids: PDGFR-{alpha}, pBS-PDGFRA (27) ; PDGFR-ß, RSV-RI (26) ; EGFR, pER1 (25) ; PKC-{delta}, pUC19-PKC-{delta} (28) ; VEGF, pBS-VEGF164 (29) ; bFGF, FGFbL2b (32) ; Met-1, pMMETS (33) ; TGF-{alpha}, pGEM-3Z-TGF-{alpha} (32) ; retinoblastoma, pMRB102 (37) ; p15INK4b, pBS-mp15INK4b (38) ; p16INK4a, pBS-mp16INK4a (38) ; Cdk4, pcMJ3/cdk4 (39) ; bax, pSFFV-mBax (40) ; Fgfr1, pFGFR1 (41) ; gli, pGEM7Z-Gli9.1 (42) ; Dep-1, pUC-DEP-1 (43) ; src, pN1.8 (44) ; p21, p21–9C (45) ; PDGF-B, pCB8B (46) ; PKC-{zeta}, pUC19-PKC-{zeta} (47) ; and PKC-{theta}, pBS-PKC-{theta} (48) .

Northern blots with 12 µg of total RNA in each lane were hybridized with these probes, and autoradiography was performed. Signals were quantified by autoradiography and densitometry and normalized to the signal obtained by hybridizing the same blots with a probe for GAPDH.


    Acknowledgments
 
Thanks to Carrie Viars and Dr. Karen Arden for help with G-banding of chromosomes. We thank the following for providing cDNA probes: Drs. Sheldon Earp, George VandeWoude, Arne Ostman, James Stone, David Lee, Gail Martin, Verne Chapman, Frederic Mushinski, Lewis Williams, Charles Sherr, Stanley Korsmeyer; Ora Bernard, Konrad Huppi, Mark Mercola, Paris Ataliotis, and Douglas Black. Also, thanks to Judson Smith for help with preparing the images in Fig. 6Citation .


    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 Supported by the California Division of the American Cancer Society, Fellowship 1-62-95 (to O. B.). Back

2 To whom requests for reprints should be addressed, at Virginia Commonwealth University, P. O. Box 980709, Richmond, VA 23298. E-mail: obogler{at}hsc.vcu.edu Back

3 The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; EGFR, EGF receptor; PKC, protein kinase C; PLAP, placental alkaline phosphatase; DMEM-bFGF, DMEM supplemented with 0.5% FCS and 10 ng/ml bFGF; DMEM-EGF, DMEM supplemented with 0.5% FCS and 20 ng/ml EGF; DMEM-FCS, DMEM supplemented with 10% FCS; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; GFAP, glial fibrillary acidic protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received for publication 9/ 4/98. Revision received 10/29/98. Accepted for publication 12/10/98.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Sidransky D., Mikkelsen T., Schwechheimer K., Rosenblum M. L., Cavenee W. K., Vogelstein B. Clonal expansion of p53 mutant cells is associated with brain tumour progression. Nature (Lond.), 355: 846-847, 1992.[Medline]
  2. Bogler O., Huang H. J., Kleihues P., Cavenee W. K. The p53 gene and its role in human brain tumors. Glia, 15: 308-327, 1995.[Medline]
  3. Reifenberger G., Liu L., Ichimura K., Schmidt E. E., Collins V. P. Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. Cancer Res., 53: 2736-2739, 1993.[Abstract/Free Full Text]
  4. Biernat W., Kleihues P., Yonekawa Y., Ohgaki H. Amplification and overexpression of MDM2 in primary (de novo) glioblastomas. J. Neuropathol. Exp. Neurol., 56: 180-185, 1997.[Medline]
  5. Watanabe K., Tachibana O., Sata K., Yonekawa Y., Kleihues P., Ohgaki H. Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas. Brain Pathol., 6: 217-223, 1996.[Medline]
  6. Chung R., Whaley J., Kley N., Anderson K., Louis D., Menon A., Hettlich C., Freiman R., Hedley-Whyte E. T., Martuza R., et al TP53 gene mutations and 17p deletions in human astrocytomas. Genes Chromosomes Cancer, 3: 323-331, 1991.[Medline]
  7. Yahanda A. M., Bruner J. M., Donehower L. A., Morrison R. S. Astrocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas. Mol. Cell. Biol., 15: 4249-4259, 1995.[Abstract/Free Full Text]
  8. Tang P., Steck P. A., Yung W. K. The autocrine loop of TGF-{alpha}/EGFR and brain tumors. J. Neurooncol., 35: 303-314, 1997.[Medline]
  9. Moscatello D. K., Montgomery R. B., Sundareshan P., McDanel H., Wong M. Y., Wong A. J. Transformational and altered signal transduction by a naturally occurring mutant EGF receptor. Oncogene, 13: 85-96, 1996.[Medline]
  10. Hunter T. Oncoprotein networks. Cell, 88: 333-346, 1997.[Medline]
  11. Noble M., Mayer-Proschel M. Growth factors, glia and gliomas. J. Neurooncol., 35: 193-209, 1997.[Medline]
  12. Bogler O., Wren D., Barnett S. C., Land H., Noble M. Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells. Proc. Natl. Acad. Sci. USA, 87: 6368-6372, 1990.[Abstract/Free Full Text]
  13. Hart I. K., Richardson W. D., Bolsover S. R., Raff M. C. PDGF and intracellular signaling in the timing of oligodendrocyte differentiation. J. Cell Biol., 109: 3411-3417, 1989.[Abstract/Free Full Text]
  14. Mayer M., Bogler O., Noble M. The inhibition of oligodendrocytic differentiation of O-2A progenitors caused by basic fibroblast growth factor is overridden by astrocytes. Glia, 8: 12-19, 1993.[Medline]
  15. Noble M., Murray K., Stroobant P., Waterfield M. D., Riddle P. Platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell. Nature (Lond.), 333: 560-562, 1988.[Medline]
  16. Vergeli M., Mazzanti B., Ballerini C., Gran B., Amaducci L., Massacesi L. Transforming growth factor-ß1 inhibits the proliferation of rat astrocytes induced by serum and growth factors. J. Neurosci. Res., 40: 127-133, 1995.[Medline]
  17. Gately S., Soff G. A., Brem S. The potential role of basic fibroblast growth factor in the transformation of cultured primary human fetal astrocytes and the proliferation of human glioma (U-87) cells. Neurosurgery, 37: 723-730, 1995.[Medline]
  18. Bogler O., Huang H. J., Cavenee W. K. Loss of wild-type p53 bestows a growth advantage on primary cortical astrocytes and facilitates their in vitro transformation. Cancer Res., 55: 2746-2751, 1995.[Abstract/Free Full Text]
  19. Eisenbarth G. S., Walsh F. S., Nirenberg M. Monoclonal antibody to plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. USA, 76: 4913-4917, 1979.[Abstract/Free Full Text]
  20. Miller R. H., Szigeti V. Clonal analysis of astrocyte diversity in neonatal rat spinal cord cultures. Development (Camb.), 113: 353-362, 1991.[Abstract]
  21. Raff M. C., Miller R. H., Noble M. A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature (Lond.), 303: 390-396, 1983.[Medline]
  22. Fukasawa K., Choi T., Kuriyama R., Rulong S., Vande W. Abnormal centrosome amplification in the absence of p53. Science (Washington DC), 271: 1744-1747, 1996.[Abstract]
  23. Agapova L. S., Ilyinskaya G. V., Turovets N. A., Ivanov A. V., Chumakov P. M., Kopnin B. P. Chromosome changes caused by alterations of p53 expression. Mutat. Res., 354: 129-138, 1996.[Medline]
  24. Fields-Berry S. C., Halliday A. L., Cepko C. L. A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. Proc. Natl. Acad. Sci. USA, 89: 693-697, 1992.[Abstract/Free Full Text]
  25. Luetteke N. C., Phillips H. K., Qiu T. H., Copeland N. G., Earp H. S., Jenkins N. A., Lee D. C. The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes Dev., 8: 399-413, 1994.[Abstract/Free Full Text]
  26. Shinbrot E., Peters K. G., Williams L. T. Expression of the platelet-derived growth factor ß receptor during organogenesis and tissue differentiation in the mouse embryo. Dev. Dyn., 199: 169-175, 1994.[Medline]
  27. Stephenson D. A., Mercola M., Anderson E., Wang C., Stiles C. D., Bowen-Pope D. F., Chapman V. M. Platelet-derived growth factor receptor a-subunit gene (Pdgfra) is deleted in the mouse patch (Ph) mutation. Proc. Natl. Acad. Sci. USA, 88: 6-10, 1991.[Abstract/Free Full Text]
  28. Mischak H., Bodenteich A., Kolch W., Goodnight J., Hofer F., Mushinski J. F. Mouse protein kinase C-{delta}, the major isoform expressed in mouse hemopoietic cells: sequence of the cDNA, expression patterns and characterization of the protein. Biochemistry, 30: 7925-7931, 1991.[Medline]
  29. Cheng S. Y., Nagane M., Huang H. S., Cavenee W. K. Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189. Proc. Natl. Acad. Sci. USA, 94: 12081-12087, 1997.[Abstract/Free Full Text]
  30. Bigner S. H., Humphrey P. A., Wong A. J., Vogelstein B., Mark J., Friedman H. S., Bigner D. D. Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res., 50: 8017-8022, 1990.[Abstract/Free Full Text]
  31. Fleming T. P., Saxena A., Clark W. C., Robertson J. T., Oldfield E. H., Aaronson S. A., Ali I. U. Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors. Cancer Res., 52: 4550-4553, 1992.[Abstract/Free Full Text]
  32. Hebert J. M., Basilico C., Goldfarb M., Haub O., Martin G. R. Isolation of cDNAs encoding four mouse FGF family members and characterization of their expression patterns during embryogenesis. Dev. Biol., 138: 454-463, 1990.[Medline]
  33. Iyer A., Kmiecik T. E., Park M., Daar I., Blair D., Dunn K. J., Sutrave P., Ihle J. N., Bodescot M., Vande W. Structure, tissue-specific expression, and transforming activity of the mouse met protooncogene. Cell Growth Differ., 1: 87-95, 1990.[Abstract]
  34. Lee D. C., Rose T. M., Webb N. R., Todaro G. J. Cloning and sequence analysis of a cDNA for rat transforming growth factor-{alpha}. Nature (Lond.), 313: 489-491, 1985.[Medline]
  35. Rong S., Donehower L. A., Hansen M. F., Strong L., Tainsky M., Jeffers M., Resau J. H., Hudson E., Tsarfaty I., VandeWoude G. Met proto-oncogene product is overexpressed in tumors of p53-deficient mice and tumors of Li-Fraumeni patients. Cancer Res., 55: 1963-1970, 1995.[Abstract/Free Full Text]
  36. Schlegel U., Moots P. L., Rosenblum M. K., Thaler H. T., Furneaux H. M. Expression of transforming growth factor {alpha} in human gliomas. Oncogene, 5: 1839-1842, 1990.[Medline]
  37. Stone J. C., Crosby J. L., Kozak C. A., Schievella A. R., Bernard R., Nadeau J. H. The murine retinoblastoma homolog maps to chromosome 14 near Es-10. Genomics, 5: 70-75, 1989.[Medline]
  38. Quelle D. E., Ashmun R. A., Hannon G. J., Rehberger P. A., Trono D., Richter K. H., Walker C., Beach D., Sherr C. J., Serrano M. Cloning and characterization of murine p16INK4a and p15INK4b genes. Oncogene, 11: 635-645, 1995.[Medline]
  39. Matsushime H., Ewen M. E., Strom D. K., Kato J. Y., Hanks S. K., Roussel M. F., Sherr C. J. Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell, 71: 323-334, 1992.[Medline]
  40. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74: 609-619, 1993.[Medline]
  41. Reid H. H., Wilks A. F., Bernard O. Two forms of the basic fibroblast growth factor receptor-like mRNA are expressed in the developing mouse brain. Proc. Natl. Acad. Sci. USA, 87: 87: 1596-1600, 1990.[Abstract/Free Full Text]
  42. Walterhouse D., Ahmed M., Slusarski D., Kalamaras J., Boucher D., Holmgren R., Iannaccone P. gli, a zinc finger transcriptional factor and oncogene, is expressed during normal mouse development. Dev. Dyn., 196: 91-102, 1993.[Medline]
  43. Ostman A., Yang Q., Tonks N. K. Expression of DEP-1, a receptor-like protein tyrosine-phosphatase, is enhanced with increasing cell density. Proc. Natl. Acad. Sci. USA, 91: 9680-9684, 1994.[Abstract/Free Full Text]
  44. Martinez R., Mathey-Prevot B., Bernards A., Baltimore D. Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart. Science (Washington DC), 237: 411-415, 1987.[Abstract/Free Full Text]
  45. Huppi K., Siwarski D., Shaughnessy J. D., Jr., Mushinski J. F. Co-amplification of c-myc/pvt-1 in immortalized mouse B-lymphocytic cell lines results in a novel pvt-1/AJ-1 transcript. Int. J. Cancer, 53: 493-498, 1993.[Medline]
  46. Mercola M., Wang C. Y., Kelly J., Brownlee C., Jackson-Grusby L., Stiles C., Bowen-Pope D. Selective expression of PDGF A and its receptor during early mouse embryogenesis. Dev. Biol., 138: 114-122, 1990.[Medline]
  47. Goodnight J., Kazanietz M. G., Blumberg P., Mushinski J. F., Mischak H. The cDNA sequence, expression pattern and protein characteristics of mouse protein kinase C-{zeta}. Gene (Amst.), 122: 305-311, 1992.[Medline]
  48. Mischak H., Goodnight J., Henderson D. W., Osada S., Ohno S., Mushinski J. F. Unique expression pattern of protein kinase C-{theta}: high mRNA levels in normal mouse testes and in T-lymphocytic cells and neoplasms. FEBS Lett., 326: 51-55, 1993.[Medline]
  49. McCarthy K. D., De Vellis J. Preparation of separate astrocyte and oligodendrocyte cultures from rat cerebral tissue. J. Cell Biol., 85: 890-902, 1980.[Abstract/Free Full Text]
  50. Harvey M., Sands A. T., Weiss R. S., Hegi M. E., Wiseman R. W., Pantazis P., Giovanella B. C., Tainsky M. A., Bradley A., Donehower L. A. In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene, 8: 2457-2467, 1993.[Medline]
  51. Santarius T., Kirsch M., Rossi M. L., Black P. M. Molecular aspects of neuro-oncology. Clin. Neurol. Neurosurg., 99: 184-195, 1997.[Medline]
  52. Bredel M., Pollack I. F. The role of protein kinase C (PKC) in the evolution and proliferation of malignant gliomas, and the application of PKC inhibition as a novel approach to anti-glioma therapy. Acta Neurochir. (Wien.), 139: 1000-1013, 1997.[Medline]
  53. James C. D., Olson J. J. Molecular genetics and molecular biology advances in brain tumors. Curr. Opin. Oncol., 8: 188-195, 1996.[Medline]
  54. Guha A., Dashner K., Black P. M., Wagner J. A. S. C. D. Expression of PDGF and PDGF receptors in human astrocytoma operation specimens supports the existence of an autocrine loop. Int. J. Cancer, 60: 168-173, 1995.[Medline]
  55. Mauro A., Bulfone A., Turco E., Schiffer D. Coexpression of platelet-derived growth factor (PDGF) B chain and PDGF B-type receptor in human gliomas. Childs Nerv. Syst., 7: 432-436, 1991.[Medline]
  56. Westermark B., Heldin C. H., Nister M. Platelet-derived growth factor in human glioma. Glia, 15: 257-263, 1995.[Medline]
  57. Hermanson M., Funa K., Koopman J., Maintz D., Waha A., Westermark B., Heldin C. H., Wiestler O. D., Louis D. N., von Deimling A., Nister M. Association of loss of heterozygosity on chromosome 17p with high platelet-derived growth factor {alpha} receptor expression in human malignant gliomas. Cancer Res., 56: 164-171, 1996.[Abstract/Free Full Text]
  58. Hermanson M., Funa K., Hartman M., Claesson-Welsh L., Heldin C. H., Westermark B., Nister M. Platelet-derived growth factor and its receptors in human glioma tissue: expression of messenger RNA and protein suggests the presence of autocrine and paracrine loops. Cancer Res., 52: 3213-3219, 1992.[Abstract/Free Full Text]
  59. Arap W., Knudsen E. S., Wang J. Y., Cavenee W. K., Huang H. J. Point mutations can inactivate in vitro and in vivo activities of p16(INK4a)/CDKN2A in human glioma. Oncogene, 14: 603-609, 1997.[Medline]
  60. Lane D. P. p53, guardian of the genome. Nature (Lond.), 358: 15-16, 1992.[Medline]
  61. Wilkin G. P., Marriott D. R., Cholewinski A. J. Astrocyte heterogeneity. Trends Neurosci., 13: 43-46, 1990.[Medline]
  62. Donehower L. A., Harvey M., Slagle B. L., McArthur M. J., Montgomery C. A., Butel J. S., Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature (Lond.), 356: 215-221, 1992.[Medline]
  63. Noble M., Murray K. Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell. EMBO J., 3: 2243-2247, 1984.[Medline]
  64. Barch M. J. The Association of Cytogenetic Technologists Cytogenetics Laboratory Manual275 Raven Press New York 1998.
  65. Lee W. H., Bister K., Moscovici C., Duesberg P. H. Temperature-sensitive mutants of Fujinami sarcoma virus: tumorigenicity and reversible phosphorylation of the transforming p140 protein. J. Virol., 38: 1064-1076, 1981.[Abstract/Free Full Text]
  66. Nagane M., Coufal F., Lin H., Bogler O., Cavenee W. K., Huang H. J. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res., 56: 5079-5086, 1996.[Abstract/Free Full Text]



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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation