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[Cancer Research 63, 1737-1742, April 15, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Invade or Proliferate? Two Contrasting Events in Malignant Behavior Governed by p16INK4a and an Intact Rb Pathway Illustrated by a Model System of Basal Cell Carcinoma1

Sofie Svensson, Kristina Nilsson, Anita Ringberg and Göran Landberg2

Departments of Laboratory Medicine, Division of Pathology [S. S., K. N., G. L.], and Plastic and Reconstructive Surgery [A. R.], Lund University, Malmö University Hospital, S-205 02 Malmö, Sweden


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
Using a highly infiltrative tumor type as basal cell carcinoma as the model system, we have examined the relation between invasive behavior and proliferation. Our results studying alterations in G1-S cell cycle regulatory proteins and proliferation in infiltrative cells were surprising and clearly indicated that invasion in tumors with an intact p16INK4a-cyclin D-retinoblastoma protein (Rb) pathway was equivalent to ceased proliferation. Using immunohistochemistry and Western blotting of microdissected parts of basal cell carcinomas, we showed that p16INK4a was up-regulated at the invasive front of the majority of basal cell carcinomas with infiltrative growth patterns, followed by ceased proliferation, as well as decreased phosphorylation of Rb. Besides supporting the fact that basal cell carcinomas have an intact Rb pathway, our results clearly indicate that invasive tumor cells change phenotype from a proliferative state to an invasive phenotype. Thus, invasion is not necessarily analogous with proliferation, implicating a paradigm shift in the understanding of two central processes in malignant behavior.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
BCC3 is the most common form of skin cancer with an occasional very aggressive and locally highly infiltrative tumor growth, despite, in general, a lack of metastasizing properties (1 , 2) . Many BCCs have high proliferative activity, as well as distinct areas with infiltrative tumor growth, making BCC a suitable model system for studies of invasion and proliferation control. Cell proliferation is regulated by a family of CDKs that are activated by cyclins, such as cyclins D and E, and inhibited by CDKIs, e.g., p27Kip1, p21Cip1, and p16INK4a (3) . The cell cycle regulator p16INK4a specifically inhibits CDK4/6 and consequently cyclin D-dependent phosphorylation of the Rb (4) , leading to less transcription of E2F-responsive genes necessary for S phase entry (5) . Besides the fact that cell cycle regulators are vital for normal proliferation control, many cell cycle regulatory gene products are further targeted in the transformation process, and the deregulation of the cell cycle is one of the key events in the development of malignancy. Regarding the potential coregulation of invasion and proliferation, there seems to exist a general assumption that invading tumor cells continue to proliferate, with only few reports stating the opposite (6 , 7) . We have earlier reported that small tumor clusters at the invasive front of colorectal cancer have lower proliferation than the tumor cells in larger tumor clusters, supporting that the more actively invading tumor cells indeed have ceased proliferation (8) . In this study, we use BCC as a model system to clarify the proliferative regulation and status of actively invading cells. Our results clearly indicate that infiltrative tumor cells do not actively proliferate and are basically in a resting G0 phase, which is in contrast to the high proliferation observed in the large bulk of tumor cells. We also show that this effect most likely is mediated through an up-regulation of the CDKI p16INK4a and executed through a functional p16INK4a-cyclin D-Rb pathway.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Tumor Materials and Immunohistochemistry.
Forty-seven sporadic occurring BCC samples were used in the study, and all diagnoses were confirmed. Tumors were further divided into three groups depending on their growth pattern and histological invasive appearance: (a) superficial; (b) nodular; and (c) BCC with infiltrative growth pattern. For immunohistochemistry, paraffin sections of 4 µm were deparaffinized using xylen and rehydrated using descending concentrations of ethanol according to standard protocol. For the antibodies antihuman p16INK4a (1:200; BD PharMingen, San Jose, CA), antihuman Ki-67 antigen (1:200; DAKO A/S, Glostrup, Denmark), and antihuman phospho-Rb (1:150; Cell Signaling Technology, Beverly, MA), antigen retrieval was achieved using 10 mM citrate buffer at pH 6.0 and microwave treatment. Single staining was performed in a DAKO Techmate 500 and all double staining in a Ventana Benchmark (Ventana Medical Systems, Inc., Tucson, AZ), according to the manufacturer’s instructions. The p16INK4a staining was confirmed with an additional antihuman p16INK4a antibody (1:100; Alexis Biochemicals, Lausen, Switzerland) using a Target retrieval solution S-3307 (DAKO; pH 8.2) at 98°C for 45 min and a DAKO Catalyzed Signal Amplification System, Peroxidase, K1500, according to the manufacturer’s instructions.

Immunohistochemical stainings were independently evaluated twice. p16INK4a-positive staining toward the epidermis (ulceration), at the nodular invasive front, and in irregularly branching strands of infiltrative tumor cells was considered (Table 1)Citation . The association between proliferation (Ki-67) and p16 was detailed in 10 BCCs using serial sections, and 9 of these BCCs were further evaluated using p16 and Ki-67 double staining. In each of these 9 sections, the number of Ki-67-positive cells per 200 p16INK4a-positive and 200 -negative cells, respectively, was denoted. The relation between Rb, Ki-67, and p16INK4a was evaluated in 12 different BCCs using serial sections. Three representative tumors were selected, and Ki-67- and Rb-positive cells were counted in three areas with high p16INK4a content and three areas with low p16INK4a content per BCC.


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  		Table 1 Results from the evaluation of the immunohistochemical straining in basal cell carcinomas

 
Western Blotting.
The MDA-MB-468 breast cancer cell line (American Type Culture Collection, Rockville, MD) used as a p16INK4a-positive control was grown in RPMI 1640, supplemented with 10% FCS, 18 units/ml penicillin, and 8 µg/ml streptomycin. Protein extracts from the cell line and tumors were prepared as described earlier (9) . Briefly, tumors were collected immediately after surgery, and areas corresponding to BCC were homogenized and sonicated for 2 x 15 s in lysis buffer, supplemented with the protease inhibitor cocktail Complete Mini (Roche, Mannheim, Germany), and kept on ice for 30 min. For electrophoresis, 30 µg of protein per lane were run on a 12% SDS-PAGE, followed by transfer to a nitrocellulose membrane Hybond ECL (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). The membranes were probed with antihuman p16INK4a (1:1000; BD PharMingen), antihuman cyclin A2 (1:350, BD Transduction Laboratories), or antihuman actin (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies, followed by peroxidase-conjugated antimouse (1:10000; Amersham Life Science, Alesbury, United Kingdom) or antigoat (1:10000; Sigma) antibodies. The proteins were detected by an ECL detection system plus (ECL+ plus) reagent (Amersham Pharmacia Biotech), according to the manufacturer’s instructions, and exposed on ECL Hyper Film (Amersham Pharmacia Biotech).

Microdissection for Western Blotting and Flow Cytometry.
Central regions of BCC samples were instantly frozen in liquid nitrogen directly after surgery, and serial sections of 8 µm, then two 200 µm, followed by an 8 µm were prepared. The 8-µm sections were stained with H&E, according to routine procedures used for sections frozen in liquid nitrogen. The 8-µm sections were used for orientation and localization of the BCC cells in the 200-µm sections. The microdissection was performed using a scalpel and dissection microscope. Areas with histological different infiltrative properties were selected. For Western blotting, the microdissected areas were boiled in loading buffer containing a final concentration of 60 mM Tris-HCl (pH 6.8), 25% (volume for volume) glycerol, 2% (w/v) SDS, 14.4 mM 2-mercaptoethanol, and 0.1% (w/v) bromophenolblue and loaded on a 12% SDS-PAGE. The proteins were electrophoretically transferred to a nitrocellulose membrane and visualized as described above. Densitometric analysis was performed with a Fluor-S quantitative imaging system (Bio-Rad Laboratories, Hercules, CA). For flow cytometry, cell suspensions were prepared from the microdissected areas by mechanically disrupting the tissue using tweezers in 3.5 µM Tris-HCl (pH 7.6), 10 mM NaCl, 10 µg/ml propidium iodide, 20 µg/ml RNase, and 0.1% (volume for volume) NP40. DNA analyses were performed in a FACSCalibur flowcytometer (Becton Dickinson Immunocytometry System, San Jose, CA), and the fraction of cells in S phase was determined manually using CellQuest 3.2 Software (Becton Dickinson Immunocytometry System).


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
Initially, p16INK4a reactivity was characterized by immunohistochemistry in 47 paraffin-embedded BCC samples with different degrees of invasive behavior, and the presence of p16INK4a staining together with the position of p16INK4a-positive tumor cells in the tumors were evaluated. Strong nuclear and cytoplasmic p16INK4a staining was found in a fraction of the BCC cells, and although cytoplasmic localization of p16INK4a has been argued (10) , the majority of cells with cytoplasmic staining was also positive in the nucleus, indicating true p16INK4a reactivity in the cytoplasm. An identical staining pattern was further obtained using another p16INK4a-reacting antibody supporting the specificity of the reactivity. Interestingly, p16INK4a reactivity was mainly seen in tumor cells located at the edges of nodules and also in tumor areas with clear infiltrative growth pattern (Fig. 1, A–C)Citation . p16INK4a reactivity was not observed in the normal keratinocytes in the epidermis nor tumor cells in the center of the BCCs, except for a few randomly scattered cells without specificity regarding the position of the cells. Presence of p16INK4a-positive tumor cells correlated further with a histologically more invasive subgroup (2) , and superficial and nodular BCCs differed substantially from BCCs with infiltrative growth patterns regarding p16INK4a expression (Table 1)Citation . These results linking p16INK4a expression to a highly invasive BCC subtype were unexpected, because deregulation of the p16INK4a gene product through loss of heterozygosity, promoter hypermethylation, and/or mutations in the INK4a/ARF locus is a common phenomenon in the transformation process, often found in more aggressive tumors (5) . Even more surprising was the regional localization of p16INK4a to obvious infiltrative parts of the BCCs. p16INK4a was not only present in the irregularly infiltrative parts of the BCCs but was often strongly expressed in tumor cells toward ulcerations in the epidermis, a common feature in some aggressive BCCs (1) .



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  		Fig. 1. Immunohistochemical staining of a BCC. A, p16INK4a staining (brown) of a nodular BCC with an infiltrative area. p16INK4a staining at the nodular invasive front (B) and in clearly infiltrative parts of the tumor (C). D–F, corresponding sections stained with Ki-67 (brown) showing ceased proliferation at the invasive front of the nodules and in clearly infiltrative parts of the BCC. G, double staining showing p16INK4a (brown) and Ki-67 (red) at the edge of the nodules. H, double staining showing p16INK4a (brown) and Ki-67 (red) in the center of the tumor.

 
The mechanism behind this up-regulation of p16INK4a in the apparently infiltrative cells is thus far unknown, and it is unclear whether p16INK4a is regulated at the transcriptional or post-transcriptional level. Candidate regulatory gene products might be the Ets family (11) of transcription factors or ß-catenin (12) . Ets-1 accumulates during senescence and can further up-regulate p16INK4a. There is also evidence that Ets-2 can induce p16INK4a expression, an effect potentiated by the Ras-Raf-mitogen-activated protein/extracellular signal-regulated kinase signaling pathway (11) . In addition, Id1 (13) , E47 (14) , and Sp1 (15) are potential regulators of the p16INK4a promoter. Besides direct transcriptional activation, it is possible that the regional p16INK4a expression can be caused by an epigenetic phenomenon, such as silencing of the INK4a promoter by hypermethylation. As supported by our results, p16INK4a seems to be functional and present in the majority of BCCs, and mutations in the INK4a/ARF locus on chromosome 9p21 are also rare in BCC (16) , although allelic loss of one or more loci on chromosome 9 has been reported (16 , 17) . The PTCH pathway, which transcriptionally represses the Wnt protein family, is commonly targeted in BCC, and in familiar forms, PTCH is inactivated (18 , 19) . The BCCs used in this study were sporadic cases, and the observed variation in p16INK4a expression suggests a regulatory event and not a constitutive activation of p16INK4a, making it less likely that PTCH is involved in the observed phenomenon. However, a recent study suggests that Wnt-1 might be associated with dedifferentiation and proliferation in BCC (20) .

p16INK4a is one of several CDKIs involved in cell cycle regulation, and we therefore screened the other members of the INK4 family, p15INK4b, p18INK4c, and p19INK4d, as well as members in the CIP/KIP family, p21cip1 and p27kip1, using immunohistochemistry. None of the additional cell cycle inhibitors showed any regional difference in expression as observed for p16INK4a. There was further no obvious regional difference for activating cell cycle regulators, such as cyclin D1, E, or A1 and CDK2, 4, or 6 (data not shown).

The result of increased p16INK4a expression in infiltrating cells was clearly unanticipated, because inactivation of p16INK4a is commonly associated with more malignant features in many tumors (3) , whereas these results instead suggested that p16INK4a was linked to invasive properties. Cell line studies have reported conflicting results regarding any potential effect of p16INK4a on invasive behavior. Restoration of p16 in the human glioma cell line SNB19 suppressed proliferation, as well as invasion (21) . In contrast, p16INK4a was up-regulated in highly infiltrative MCF-7 breast cancer cells transfected with c-Jun supporting the results presented here (22) .

To investigate whether the cells with an up-regulated p16INK4a were proliferating and if the CDK4/6-cyclin D-Rb pathway was intact in BCC, we used the proliferation marker Ki-67. The results clearly showed a decrease in proliferation in the p16INK4a high areas (Fig. 1 D–F)Citation , indicating the existence of a functional p16INK4a-cyclin D-Rb pathway in BCCs. To further validate the Rb function in BCC, we used an antibody specifically detecting phosphorylation of Rb serines (Ser807 and Ser811). These residues are phosphorylated by the cyclin D1-CDK4 complex (23) . Immunohistochemical analyses of p16INK4a high areas showed a subsequent decrease in Rb phosphorylation, similar to the proliferation decrease, verifying the existence of a functional Rb and an effect of p16INK4a on the phosphorylation of the key G1-S regulatory substrate Rb (Fig. 2)Citation . A high fraction of Rb phosphorylated cells was accordingly observed in the center of the tumor with no detectable p16INK4a.



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  		Fig. 2. Phospho-Rb- and Ki-67-positive tumor cells in p16INK4a-high and -low areas. Histogram showing the fraction (mean ± SD) phospho-Rb- and Ki-67-positive tumor cells in p16INK4a-high and -low areas in three representative BCCs. The immunohistochemical staining shows phospho-Rb (brown) in a p16INK4a-high and -low area.

 
These results suggest that there is a phenotypic change from a highly proliferative cell in the center of the tumor mass toward a cell with lower proliferation and infiltrative behavior at the edge of the tumor. The question is if this finding in BCC is of general importance and applicable to other malignancies. We and others have earlier reported a similar phenomenon in colorectal cancer, where increased p16INK4a and decreased proliferation were detected in small invasive tumor clusters but not in larger tumor clusters (8 , 12) . In colorectal tumors lacking functional p16INK4a or Rb, there was nevertheless sustained proliferation in infiltrative clusters (8) . Similar observations have also been made in human glioma cell lines, where migrating cells had lower proliferation, although the underlying mechanism of this decrease in proliferation was not clear (7) . Decreased proliferation in invading tumor cells caused by p16INK4a up-regulation could therefore potentially be a general event in malignancies but with a requirement of a functional p16INK4a-cyclin D-Rb pathway. The results presented here using BCC as a model system are nevertheless the most definite thus far, and the inverse association between p16INK4a and proliferation and the unambiguous p16INK4a up-regulation in infiltrative cells is exceptionally clear.

To verify the results obtained from the immunohistochemical analyses of BCC, we performed Western blotting of protein extracts prepared from 12 fresh BCCs and nontumor tissues. As illustrated (Fig. 3A)Citation , a band corresponding to p16INK4a was observed in several of the BCC samples, as well as in the positive control cell line MDA-MB-468, whereas normal skin and some BCC samples were negative. To further confirm that p16INK4a is up-regulated at the invasive margin and the association to ceased proliferation, we froze BCCs in liquid nitrogen and used microdissection, followed by Western blotting and flow cytometry (in a subset of tumors), to verify the regional differences. The dissected parts were divided into different areas depending on their invasive appearance. The proliferative activity was monitored by cyclin A2 and flow cytometric S phase analyses. There was an inverse association between p16INK4a and cyclin A2 in the microdissected areas (Fig. 3, C and E)Citation further corresponding to the immunohistochemical analyses of the same tumor (Fig. 3B)Citation . The flow cytometric analyses of microdissected areas also showed a variation in S phase fractions (range, 0.8–5.2%) between different parts of the tumor, mirroring the cyclin A2 data (Fig. 3, D and E)Citation . The dissected BCC areas considered to be more infiltrative had accordingly higher amounts of p16INK4a and lower proliferative activity. This confirms our immunohistochemical findings of a decreased proliferative activity at the invasive front most likely caused by an up-regulation of p16INK4a.



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  		Fig. 3. BCCs and microdissected areas analyzed by Western blotting and flow cytometry. A, BCC protein extracts analyzed by Western blotting illustrating p16INK4a protein expression in different tumors. Normal skin was used as negative control, and the cell line MDA-MB-468 was used as positive control. Actin was used as loading control. B, immunohistohemical p16INK4a and Ki-67 staining of the microdissected areas of BCC 18. C, Western blotting of microdissected BCC 18 showing inverse relationship between cyclin A2 (proliferation) and p16INK4a in areas with different infiltrative properties. Actin was used as loading control. D, DNA histogram from a microdissected area of BCC 37. E, relative changes in p16INK4a (white bars) and cyclin A2 (black bars) protein contents in different tumor areas of BCC 37, 18, and 32 determined by microdissection and Western blotting. The relative change in S phase (gray bars) in microdissected parts of BCC 37 was analyzed using flow cytometry. The highest densitometric value in each blot and S phase measurement were denoted a value of 1 and used for calculation of the relative changes.

 
In conclusion, our results clearly indicate that invasive BCC cells can change phenotype from a proliferative to an invasive type with low proliferative activity and that this effect most likely is mediated by p16INK4a and a functional Rb pathway. Thus, invasion and proliferation might be considered as two contrasting events in malignant behavior. The results could further explain why p16INK4a inactivation is frequent in the malignant transformation process, potentially causing simultaneous proliferation and invasion, a feature that most likely is of selective advantage in tumor progression.


   ACKNOWLEDGMENTS
 
We thank Elise Nilsson and the personnel at the Department of Plastic and Reconstructive Surgery, Malmö University Hospital, for their excellent technical assistance and the BCC patients who participated in this study.


   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 grants from the Swedish Cancer Society, Gunnar, Arvid and Elisabeth Nilsson Cancer Foundation, Lund University Research Funds, and Malmö University Hospital Research and Cancer Funds. Back

2 To whom requests for reprints should be addressed, at Department of Laboratory Medicine, Division of Pathology, Lund University, Malmö University Hospital, S-205 02 Malmö, Sweden. Fax: 46-40-337063; E-mail: goran.landberg{at}pat.mas.lu.se Back

3 The abbreviations used are: BCC, basal cell carcinoma; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; Rb, retinoblastoma protein; ECL, enhanced chemiluminescence. Back

Received 10/17/02. Accepted 2/27/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

  1. Jacobs G. H., Rippey J. J., Altini M. Prediction of aggressive behavior in basal cell carcinoma. Cancer (Phila.), 49: 533-537, 1982.[Medline]
  2. Rippey J. J. Why classify basal cell carcinomas?. Histopathology, 3: 393-398, 1998.
  3. Sherr C. J. Cancer cell cycles. Science (Wash. DC), 274: 1672-1677, 1996.[Abstract/Free Full Text]
  4. Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature (Lond.), 366: 704-707, 1993.[Medline]
  5. Hanahan D., Weinberg R. A. The hallmarks of cancer. Cell, 100: 57-70, 2000.[Medline]
  6. Bolteus A. J., Berens M. E., Pilkington G. J. Migration and invasion in brain neoplasms. Curr. Neurol. Neurosci. Rep., 1: 225-232, 2001.[Medline]
  7. Koochekpour S., Merzak A., Pilkington G. J. Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma cell lines. Eur. J. Cancer, 31A: 375-380, 1995.
  8. Palmqvist R., Rutegård J. N., Bozoky B., Landberg G., Stenling R. Human colorectal cancers with an intact p16/cyclin D1/pRb pathway have up-regulated p16 expression and decreased proliferation in small invasive tumor clusters. Am. J. Pathol., 157: 1947-1953, 2000.[Abstract/Free Full Text]
  9. Nielsen N. H., Emdin S. O., Cajander J., Landberg G. Deregulation of cyclin E and cyclin D1 in breast cancer is associated with inactivation of retinoblastoma protein. Oncogene, 14: 295-304, 1997.[Medline]
  10. Geradts J., Hruban R. H., Schutte M., Kern S. E., Maynard R. Immunohistochemical p16INK4a analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene. A comparison of four commercial antibodies. Appl. Immunohistochem. Mol. Morphol., 8: 71-79, 2000.[Medline]
  11. Ohtani N., Zebedee Z., Hout T. J. G., Stinson J. A., Sugimoto M., Ohashi Y., Sharrocks A. D., Peters G., Hara E. Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence. Nature (Lond.), 409: 1067-1070, 2000.
  12. Jung A., Schrauder M., Oswald U., Knoll C., Sellberg P., Palmqvist R., Niedobitek G., Brabletz T., Kirchner T. The invasion front of human colorectal adenocarcinomas shows co-localization of nuclear ß-catenin, cyclin D1, and p16INK4a and is a region of low proliferation. Am. J. Pathol., 159: 1613-1617, 2001.[Abstract/Free Full Text]
  13. Alani R. M., Young A. Z., Shifflett C. B. Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a. Proc. Natl. Acad. Sci., USA, 98: 7812-7816, 2001.[Abstract/Free Full Text]
  14. Pagliuca A., Gallo P., De Luca P., Lania L. Class A helix-loop-helix proteins are positive regulators of several cyclin-dependent kinase inhibitors’ promoter activity and negatively affect cell growth. Cancer Res., 60: 1376-1382, 2000.[Medline]
  15. Pagliuca A., Gallo P., Lania L. Differential role for the Sp1/Sp3 transcription factors in the regulation of the promoter activity of multiple cyclin-dependent kinase inhibitor genes. J. Cell. Biochem., 76: 360-367, 2000.[Medline]
  16. Saridaki Z., Koumantaki E., Liloglou T., Sourvinos G., Papadopoulos O., Zoras O., Spandidos D. A. High frequency of loss of heterozygosity on chromosome region 9p21–p22 but lack of p16INK4a/p19ARF mutations in greek patients with basal cell carcinoma of the skin. J. Investig. Dermatol., 115: 719-725, 2000.[Medline]
  17. Quinn A. G., Campbell C., Healy E., Rees J. L. Chromosome 9 allele loss occurs in both basal and squamous cell carcinomas of the skin. J. Investig. Dermatol., 102: 300-303, 1994.[Medline]
  18. Dahmane N., Lee J., Robins P., Heller P., Ruiz I Altaba A. Activation of the transcriptionfactor Gli1 and the sonic hedgehog pathway in skin tumors. Nature, 398: 876-881, 1997.
  19. Unden A. B., Holmberg E., Lundh-Rozell B., Ståhle-Bäckdahl M., Zaphiropoulos P. G., Toftgård R., Vorechovsky I. Mutations in the human homologue of Drosophila patched (PTCH) in basal cell carcinoma and in the Gorlin syndrome: different in vivo mechanisms of PTCH inactivation. Cancer Res., 56: 4562-4565, 1996.[Abstract/Free Full Text]
  20. Lo Muzio L., Pannone G., Staibano S., Mignogna M. D., Grieco M., Ramires P., Romito A. M., De Rosa G., Piattelli A. WNT-1 expression in basal cell carcinoma of head and neck. An immunohistochemical and confocal study with regard to the intracellular distribution of beta-catenin. Anticancer Res., 22: 565-567, 2002.[Medline]
  21. Adachi Y., Chandrasekar N., Kin Y., Lakka S. S., Mohanam S., Yanamandra N., Mohan P. M., Fuller G. N., Fang B., Fueyo J., Dinh D. H., Olivero W. C., Tamiya T., Ohmoto T., Kyritsis A. P., Rao J. S. Supression of glioma invasion and growth by adenovirus-mediated delivery of a bicistronic construct containing antisense uPAR and sense p16 gene sequences. Oncogene, 21: 87-95, 2002.[Medline]
  22. Rinehart-Kim J., Johnston M., Birrer M., Bos T. Alterations in the gene expression profile of MCF-7 breast tumor cells in response to c-Jun. Int. J. Cancer, 88: 180-190, 2000.[Medline]
  23. Zarkowska T., Mittnacht S. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J. Biol. Chem., 272: 12738-12746, 1997.[Abstract/Free Full Text]



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