H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents.
BACKGROUND
Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.


RESULTS
Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.


CONCLUSIONS
The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.
Vaughan
Melville B
MB
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America. mvaughan4@uco.edu
Ramirez
Ruben D
RD
Andrews
Capri M
CM
Wright
Woodring E
WE
Shay
Jerry W
JW
D015820:Cadherins / Q000378:metabolism; D049109:Cell Proliferation; D003094:Collagen / Q000737:chemistry; D018450:Disease Progression; D004848:Epithelium / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D009361:Neoplasm Invasiveness; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis*; D012867:Skin / Q000166:cytology*; D012878:Skin Neoplasms / Q000378:metabolism
D015820:Cadherins; D003094:Collagen; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)