Modeling of multi-step oral carcinogenesis in vitro : assessment of growth, differentiation and apoptosis markers

Human oral mucosa, especially the buccal epithelium, is worldwide a common site for cancer. Cancer development frequently results in inactivation of tumor suppressor p53, a central regulator of growth and programmed cell death, and deregulated expression of structural elements like cytokeratins. The overall aim of this study was to investigate if the multi-step process of carcinogenesis can be modeled and studied from a mechanistic stand point utilizing cultured normal (NOK), immortalized (SVpgC2a) and malignant (SqCC/Y1) human buccal keratinocytes.

Organotypic epithelia of the respective cell lines, as derived from serum-free culture on a collagen gel containing oral fibroblasts, showed morphological features ranging from normal tissue to carcinoma in situ. The respective epithelia showed sharp differences in immunochemical expression of keratins. NOK expressed many of the same keratins as buccal mucosa, whereas loss of keratins in SVpgC2a and their retention in SqCC/Y1 showed similarities to oral dysplasia and well-differentiated squamous cell carcinoma. Assessment of tissue homeostatic functions demonstrated that NOK exhibited a terminal squamous differentiation (TSD) and apoptosis- capable phenotype, that responded to fibroblast-mediated proliferation with increased apoptosis and to elevation of Ca 2+ by induction of TSD. In contrast, SVpgC2a and SqCC/Y1 exhibited hyper- proliferative, TSD-deficient and hyperapoptotic phenotypes that failed to respond to the above stimuli. Immunochemical expression of tumor suppressor p53 was scattered in NOK, heterogeneous in SVpgC2a and negative in SqCC/Y1. Exclusively for NOK, p53 expression increased with proliferation and decreased with TSD, moreover, expression of Bax, a gene associated to apoptosis in many cell types, correlated with TSD.

Further evaluation of NOK and SVpgC2a in various conditions for up to 17 days consistently showed several-fold higher proliferation and apoptosis rates in SVpgC2a. Micro-array analysis of NOK and SVpgC2a in monolayer culture confirmed the respective keratin protein profiles to the mRNA level, and indicated expression of keratins not previously reported for buccal epithelium. Under sparse or confluent culture, SVpgC2a exhibited relatively higher cloning ability and growth rate as well as lower responsiveness to contact inhibition than NOK. Apoptosis and TSD were regulated in NOK in response to increasing cell density whereas SVpgC2a showed resistance. Cultures of NOK showed obligatory dependence for the growth supplement, pituitary extract, whereas SVpgC2a showed independence, and thus, SVpgC2a could be cultured at chemically defined conditions. Immunochemical assessments in NOK showed increased Bax expression under conditions that increase TSD and decrease apoptosis, providing ftirther evidence for the dissociation of Bax expression from apoptosis in keratinocytes.

A composite in vitro model for malignant transformation of oral epithelium is described. Characterization of NOK, SVpgC2a and SqCC/Y1 demonstrated that the multi-step process of malignant transformation of buccal keratinocytes clearly associates with alterations in basic cellular functions and mechanisms that regulate tissue homeostasis and build-up of the cytoskeleton. Overall, standardized, highly defined culture conditions, different cell densities and co-culture models provide useful means of investigating mechanisms underlying oral cancer development.

List of scientific papers

I. Hansson A, Bloor BK, Haig Y, Morgan PR, Ekstrand J, Grafstrom RC (2001). Expression of keratins in normal, immortalized and malignant oral epithelia in organotypic culture. Oral Oncol. 37(5): 419-30.
https://pubmed.ncbi.nlm.nih.gov/11377230

II. Hansson A, Bloor BK, Sarang Z, Haig Y, Morgan PR, Stark HJ, Fusenig NE, Ekstrand J, Grafstrom RC (2003). Analysis of proliferation, apoptosis and keratin expression in cultured normal and immortalized human buccal keratinocytes. Eur J Oral Sci. 111(1): 34-41.
https://pubmed.ncbi.nlm.nih.gov/12558806

III. Hansson A, Zheng X, Haig Y, Bloor BK, Morgan PR, Stark HJ, Fusenig NE, Elfwing A, Grafstrom RC (2003). Growth, programmed cell death and gene expression related to p53 function in epithelia regenerated with cultured normal, immortalized and malignant human buccal keratinocytes. [Manuscript]

IV. Haig Y, Hansson A, Zheng X, Bloor BK, Morgan PR, Elfwing A, Grafstrom RC (2003). Growth, programmed cell death and gene expression related to p53 function in normal and SVT40T antigen-immortalized human buccal keratinocytes. [Manuscript]