|Budget Amount *help
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1991: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
A protein-free cell culture system for human squamous cell carcinoma of the oral cavity has been established. Using this protein-free culture system, it was revealed that human gingival carcinoma derived cell line Ca9-22PF, which has been cultured with a protein-free synthetic medium PF86-1, produced IL-1 and PDGF-AA constitutively. However, it seems to be very hard to screen out the many kinds of cytokines produced by oral cancer cells only using a proteinfree culture system. Therefore, in this study we tried to detect the expression of gene for cytokines by the polymerase chain reaction (PCR) method.
Cytoplasmic RNA was prepared from ZAPF cells, which was established from a metastatic lymph node of a palatal cancer and cultured with PF86-1, using Nonidet P-40. Further purification of mRNA was carried out by loading it on a oligo-dT cellulose column. Single strand cDNAs were synthesized with MO-MLV reverse transcriptase from this MRNA used as a template, then targeted DNA for cytokines, G-CSF, M-CSF, GM-CSF, IL-3 and IL-6 respectively, was amplified, if it had existed in the sample, by PCR method and separated by agarose gel electrophoresis (AGE).
As results, no predicted band was observed on AGE, when PCR was performed for G-CSF, IL-3 and IL-6. On the other hand, in the case of G-CSF 630 base pairs (bp) band, which was predicted from the sequence of cDNA, was visualized on AGE. In the case of M-CSF, a predicted 450 bp band was detected and it was separated to two bands of 150bp and 300bp prospected from the cDNA sequence after the digestion with Mung Bean Nulease and Stu I restriction endonuclease.
These results show that ZAPF expresses at least two cytokine genes, GM-CSF and M-CSF, however it will be necessary to investigate whether ZAPF produce GMCSF and M-CSF at biologically responsible concentration or not using a proteinfree culture system.