David J. Winterbourne

Heparan sulphate is a potent inhibitor of DNA synthesis in vitro

Summary The inhibition by glycosaminoglycans of DNA synthesis in vitro has been studied. The marked inhibition caused by heparin was reduced by chemical modification: analogues containing either N-sulphate or O-sulphate groups alone were still inhibitory, those containing either no sulphate groups or free amino groups were not. Of the widely distributed glycosaminoglycans only heparan sulphate was a strong inhibitor of DNA synthesis. The results indicate that high inhibitory activity to DNA polymerase, depends on specific structural features of the heparin family of glycosaminoglycans and is not simply related to gross charge density, although this plays a role.

Binding of heparan sulphate and heparin to control and virus-transformed cells.

A cloned embryonic mouse cell line contained specific cell-surface receptors for heparin and both the number and affinity appeared to be unchanged in a simian-virus-40-transformed subclone. In competitive binding assays heparan sulphate from the control clone was bound preferentially compared to that from the transformed subclone, indicating that the altered sulphation of heparan sulphate from transformed cells results in a lowered affinity for cell-surface receptors. Evidence was obtained suggesting that endogenous proteoglycans were not held at the cell surface by binding to these receptors alone. However the possibility that proteoglycans embedded in the plasma membrane may interact with the receptor has not been ruled out.

Human tumour cell lines established in vitro from tumours after long-term passage as nude mouse xenografts. Comparative fingerprinting of their concanavalin-A acceptor glycoproteins.

Two human colon cancer xenografts (EC and AC) were established in tissue culture only after long-term passage in nude mice. Earlier attempts to establish cell lines were unsuccessful. The epithelioid cells retain their tumourigenicity after in vitro growth, giving rise to tumours with a take rate of 60-80%. After reimplantation, the xenografts retain a similar morphology to that of the original human tumours. Both cell lines show human karyology. Comparative mapping of Concanavalin-A acceptor glycoproteins provides a fingerprint characteristic of each cell line. These glycoprotein patterns are similar to those shown by HT-29, an established colon cancer cell line.

SUPPRESSION OF ANCHORAGE-INDEPENDENT GROWTH AFTER GENE TRANSFECTION

A novel procedure for isolating anchorage-dependent cells has been developed. It involves negative selection of cells growing in suspension followed by clonal replica screening for anchorage-dependent growth. Cells which have regained anchorage-dependent growth have been isolated from a library of the Chinese hamster ovary cell line, CHO-K1, transfected with pSV2neo and human genomic DNA. One anchorage-dependent clone, 1042AC, has been studied in detail. Anchorage-dependent growth of 1042AC is stable when cultured as adherent monolayers, but revertants appear rapidly when cultured in suspension. Suppression is unlikely to be due to loss or mutation of hamster genes conferring anchorage-independent growth as hybrids between 1042AC and CHO-K1 have the suppressed phenotype of 1042AC. Furthermore, a population of cells obtained from the hybrid by selecting for revertants to anchorage-independent growth showed selective loss of the transgenome derived from 1042AC. The growth suppression was not due to transfection of the human Krev-1 gene, which has previously been shown to restore anchorage-dependent growth, nor was there any evidence of alteration in the endogenous hamster Krev-1 gene. However, evidence for a human gene being responsible for the suppressed phenotype has not been obtained yet.