Selection and Characterisation of Transformed BHK21 Cells Altered in Response to Fibronectin

Hameed, Abud al Hadi M (1988) Selection and Characterisation of Transformed BHK21 Cells Altered in Response to Fibronectin. PhD thesis, University of Glasgow.

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Abstract

I found that selection of cell lines which do not respond to fibronectin was easy from polyoma transformed cells, but yielded no such cell lines from Schmidt-Ruppin transformed cells. Mixing of Py3 mutants (F2) with SR-WT cells showed that Py3 mutants could be recovered when present at about 1:10 6 and higher inputs, while the frequency of SR mutants was zero. Cells with a genetic marker (resistance to 6-thioguanine) were isolated from polyoma transformed cells. Two colonies were selected (TG1, TG2). From one of these lines (TG2), two different low adherent lines both deficient in response to fibronectin, were selected and recloned on soft agar. Variant 1 (TG2F1) has compact colonies of rounded cells while variant 2 (TG2F2) has scattered colonies with a few slightly spread cells. Both are resistant to 6-thioguanine and have undetectable HGPRT-ase activity. Revertants of two different morphologies were isolated from the compact colony Py3 mutant (F2). Variant 1 (F2R1) has well spread cells while cells of variant 2 (F2R2) are less well spread. Both revertants were found to attach and spread on fibronectin-coated surfaces. A third type of revertant has been observed several times, which had extremely well spread cells, better spread than wild type. Attempts to isolate this type of revertant were unsuccessful. TG mutants (TG2F1 & TG2F2) were found to spread on Con A but not on WGA-coated surfaces. On poly L-lysine, both wild type and mutants adhere, but none of them spread. Wild type but not mutants spread fully on poly L-lysine in presence of 1% serum. Mutants did not spread on serum or fibronectin. Mn2+ ions were effective in inducing the spreading of TG2-WT and both variants. 10 -6 M Mn2+ was 50% effective in inducing the spreading of TG-WT on fibronectin while 10 -3 M was 50% effective on haemoglobin. Higher concentrations such as 10 -2 M were required for TG2F1 cells on both surfaces. 10 -4 M was effective with TG2F2 cells on fibronectin while 10 -3 M was effective on haemoglobin. Mg2+ at 10 -2 M was as effective as Mn2+ with parental cells on fibronectin but not as effective as Mn2+ with variants on either surfaces. Co2+ was less effective at 10 -2 M with all cell lines compared to Mn 2+ or Mg2+. Analysis of lectin binding proteins associated with the detergent soluble extracts of Py3 (NaDOC-solubilised membranes and Triton extracts) failed to reveal differences between wild type, mutants and revertants. The molecular basis for the differences between wild type and mutants could be in the fibronectin receptor, or in signal transduction required to induce the spreading response, but remains unidentified.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Cellular biology
Date of Award: 1988
Depositing User: Enlighten Team
Unique ID: glathesis:1988-77739
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53
URI: http://theses.gla.ac.uk/id/eprint/77739

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