CODEC

The pre-Bn lines Blin-1 and Nalm-6 appeared to be genetically identical when analysed by Southern blotting using the probes pXG3 and IM S 8. It appears that the line we were given under the name Blin-1 was actually Nalm-6, but because the lines had different Ca^^ signalling properties and some phenotypic differences the data obtained from Blin-1 has been reported in this thesis as an example of a pre-BII cell line that has been

1E8 could not be a sub-line derived by in vitro differentiation of Blin-1. Therefore, Blin-1 and 1E8 could not be used as a model for pre-BII to B cell differentiation. However, line 1E8 could be exploited as an example of an immature B cell line with some features in common with pre-BII cells, so data jhom this line is included in the thesis.

Lines REH and Nalm-1 also shared an identical hybridisation pattern with the probes pXG3 and XMS8. However, the phenotypes were different in that Nalm-1 was a pre-BI cell expressing cytoplasmic p protein and REH a pro-B cell line with no cytoplasmic p. For this reason, data from both lines have been retained even though the line

nomenclature is probably incorrect and my results cannot be compared directly with data published by others on these cell lines.

It is possible that many cell lines that are circulated around the world between different laboratories are subject to mix-ups like these, as it is common to accept that a line is what it is claimed to be and it is quite uncommon to subject lines to DNA fingerprinting techniques. Human error can lead to vials of cell lines being mislabelled and

contamination of a cell line with a different cell line can result in replacement of one line by another.

IVB.3.7 Signalling through the |li\|/LC complex on pre-BII

cell lines

The work in this thesis demonstrates that the pij/LC complex can transduce a signal, at least on cell lines of leukaemic origin. Lines 697 and Nalm-6 mobilised intracellular Ca2+

on ligation of the iliv|/LC complex by p-specific antibody, although Blin-1 did not. It has been reported previously that Nalm-6 makes only a weak Ca^^ response on ligation of p chain However, it has not been shown previously that a pre-BII cell line can generate a Ca^^ flux of equal amplitude to B cell lines on ligation o f p heavy chain, as was the case for line 697. The weaker Ca^^ flux of Nalm-6, which was of low amplitude and had a late peak, and the absence of Ca^^ mobilisation by Blin-1 in response to ligation of p heavy chain may be due to the absence of CD45 which is important for signalling through the B cell receptor CD45 probably dephosphorylates the negatively regulatory tyrosine residue at the C-termini of Src family tyrosine kinases and thereby activates them, which

constitutes an early signalling event through the BCR Loss of CD45 blocks BCR

signalling in the CD45 knockout mouse

Lines 697 and Blin-1 responded to ligation of p chain with an increase in protein tyrosine kinase activity, albeit a weak response involving few substrate bands. Line 697 generally tyrosine phosphorylated 4 protein bands of approximate molecular weights 112, 85, 66 and 60 kD and Blin-1 phosphorylated 2-3 bands. Nalm-6 did not give a convincing response in 8 western blotting experiments, although it did appear to respond by the method where cells were stimulated with beads coated with p-specific antibody and phosphotyrosine-specific antibody staining revealed local tyrosine kinase activity at the cell membrane when cells were viewed by fluorescent microscopy. There is some discrepancy between the results of different groups about whether human pre-BII cell lines can respond to ligation of p heavy chain with an increase in tyrosine kinase activity, probably because the response is very weak compared to the response of B cell lines so is

often missed. Bossy et al. and Kuwahara et al. report a tyrosine kinase response fi’om Nalm-6, but Nakamura et al. found no such response fi'om line 697.

In agreement with the data of other groups, the tyrosine kinase responses of pre-BII cells were weaker than those of B cell lines, involving a lower overall level of tyrosine

phosphorylation and fewer substrate bands There are therefore quantitative

differences between pre-B and B cell signal transduction through the p heavy chain which may have resulted from the lower expression density of p chain on the surface of pre-B cells or could reflect the use of different signalling pathways. Some tyrosine kinase substrates appeared to be B cell-specific and therefore would constitute evidence that the pre-BCR and the BCR also display qualitative differences in their signal transduction pathways. It is however possible that the same substrates were tyrosine phosphorylated in pre-Bn cells but were undetectable on the gels due to the lower abundance of these proteins in pre-B cells or the lower abundance of surface-bound p chain. Pre-B cell- specific substrates would provide more compelling preliminary evidence for qualitative differences between pre-BCR and BCR signalling. The functions of IgM on a B cell and the pn/LC complex on a pre-BII cell are different, so it would not be surprising if signal transduction by p heavy chain differed between these developmental stages. There are some differences in the molecules associated with the pre-BCR and the BCR, but it is not

known whether these would lead to different signalling pathways However, only

the most proximal signalling events in pyLC complex signalling have been investigated so far. A comparison of the pre-BCR on Nalm-6 and the BCR on Ramos revealed tyrosine kinase and serine/ threonine kinase activities associated with each A similar

comparison of p-associated molecules in 2 human pre-BII cell lines and 2 B cell lines showed that Lyn and Syk were present in equivalent amounts in the lines PI3 kinase was present in greater amounts in the pre-B lines, but specific inhibition of PI3 kinase with wortmannin did not affect the tyrosine phosphorylation pattern of the lines,

indicating that PI3 kinase was not responsible for the differences in early signalling events observed between pre-B and B cells. PI3 kinase may of course have a key role in pre-B cell signalling downstream of these early phosphorylation events. Ig-a and Ig-p are glycosylated differently at the various stages of human B cell differentiation which could couple them to different signalling molecules at successive developmental stages

The tyrosine kinase substrate hands were not identified in my experiments. This could have been addressed by using specific antibodies to identify substrates. Some possible candidate substrates are p i 20^^' the p i 10 and p85 subunits of PI3 kinase, the p65

Wiskott Aldrich Syndrome protein and any of the lymphocyte Src family tyrosine

kinases which have molecular weights of 56-60 kD.

It was hoped that the prospect of a switch in signalling mechanisms as a pre-BII cell matures to the B cell stage could have been addressed using Blin-1 and 1E8. The demonstration that our Blin-1 sub-line was not of the same genetic origin as 1E8 meant that this model was invalid. However, line 1E8 was a B cell line with some features of pre-B cells such as cytoplasmic expression of the v|/LC, and the tyrosine kinase response of line 1E8 on ligation of p chain was intermediate between the responses of pre-BII and B cell lines. More substrate species appeared to have been tyrosine phosphorylated in 1E8

cells than the pre-BII cell lines but the strength of the signal was weaker than in B cell lines, in agreement with Bossy et al. Contrary to their observation that 1E8 gave only a weak Ca^^ response to ligation of p chain, I found this response to be of equal amplitude to that of the B cell lines. Line 1E8 therefore provided further support for at least a quantitative difference in signalling through p heavy chain between pre-BII and immature B cells.

Cell lines cannot of course be assumed to have the same biology as normal bone marrow cells and one must be cautious not to over-interpret results which were obtained with cells that are after all aberrant. The phenotyping data are evidence enough that differences exist between cell lines and normal cells. It is probable however that if the pvj/LC complex can transduce a signal on some pre-BII cell lines then the same biochemistry and the same function exists in normal cells expressing the pre-BCR. I attempted to address this issue by investigating tyrosine kinase signalling in normal bone marrow cells, although it did not prove possible to investigate pre-B cell signalling in isolation from B cell signalling by the methods that I tried (section IHA).