Date of Award

10-1-1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy and Cell Biology

Abstract

Penetration of blood vessel basement membranes (BMs) by metastatic cells apparently proceeds by a three step process involving attachment, degradation, and migration. In the current study, isolated glomerular BMs (GBMs) were used as model substrates for studying cell attachment to BMs.Murine and bovine gomeruli were isolated by differential sieving of homogenized renal tissues. GBMs were prepared by treating glomeruli with detergents and enzymes. This was facilitated by using "sieving chambers" which prevented tissue loss during the detergent extraction procedure. The ultrastructure of detergent isolated murine and bovine GBMs was similar to other species, although they had a greater tendency to collapse.Murine melanoma cells were incubated at 37$\sp\circ$C with the following substrates for one half hour to seven days: (1) murine glomeruli, (2) murine GBMs, (3) bovine GBMs, and (4) BM protein coated tissue culture wells. Normal mouse testis (NMT) cells were similarly incubated with the following substrates: (1) bovine GBMs, and (2) BM protein coated wells. Electron microscopic analysis showed that following co-incubation, K-1735-M2 (M2) melanoma cells attached to both murine glomeruli and GBMs but podocytes appeared to interfere with attachment to glomeruli. Both NMT and melanoma cells attached to bovine GBMs. No GBM degradation was observed in either species. Quantitative assays using radiolabeled cells showed that melanoma cells attached to bovine GBMs in greater numbers than NMT cells. There was no difference, however, in the attachment to bovine GBMs of highly metastatic M2 and low metastatic K-1735-16 cells. NMT cell attachment to laminin coated wells was greater, and M2 cell attachment less, than that described by others. Furthermore, treatment of M2 cells with laminin or fibronectin had almost no effect on attachment except that laminin treatment decreased M2 cell attachment to fibronectin coated wells.These results demonstrate that isolated bovine GBMs can be used as model substrates for investigating cell attachment to BMs. Furthermore, while attachment appears essential for metastasis, use of this model in the present study suggests that attachment rates may not determine in vivo metastatic potential. Finally, in contrast to other melanoma cell lines, the major mediator of M2 cell attachment appears to be fibronectin rather than laminin.

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