Abstract
Laminin promotes the malignant phenotype, and the expression of certain laminin receptors is increased in malignancy. Previously, we demonstrated that a laminin-adhesive subclone of a human colon cancer cell line showed increased tumorigenicity in nude mice and increased affinity of the beta1 integrin for laminin relative to the laminin-non-adhesive subclone. The total amount of either beta1 integrin protein or mRNA did not increase. As levels of the 32/67-kDa laminin receptor (67LR) correlate with malignancy, we examined 67LR expression in the laminin adhesion-selected human colon cancer cells. The laminin-adhesive subclone, which was more tumorigenic in both heterotopic and orthotopic locations than in a laminin-non-adhesive subclone, showed cell-surface membrane staining of 67LR, whereas the laminin-non-adhesive subclone showed cytoplasmic staining of 67LR. No difference in either the amount of 67LR mRNA or the amount of protein was observed in the parental cells than in the laminin-adhesive and non-adhesive subclones. When assayed on a laminin affinity column, more 67LR molecules bound to the column with cell extracts from the laminin-adhesive subclone than was observed with the non-adhesive subclone. These findings suggest that the increased tumorigenicity of laminin adhesion-selected tumour cells might be due to an alteration in the distribution and/or adhesiveness of multiple receptors including 67LR and beta1 integrin.
Highlights
The results showed that differences in tumorigenicity might be caused by alterations in multiple laminin receptors, including R1 integrin and 67LR
Laminin and Matrigel were prepared from the EngelbrethHolm-Swarm (EHS) tumour, which is known to produce a large amount of basement membrane
The ET+ subclone showed the strongest attachment to laminin at concentrations higher than 1 jg per well in the 96-well plate
Summary
Cell culture and selectionLaminin and Matrigel were prepared from the EngelbrethHolm-Swarm (EHS) tumour, which is known to produce a large amount of basement membrane. Antibody against 110 kDa laminin receptor was prepared using purified protein from fetal mouse brain (Kleinman et al, 1991). A biopsy specimen from a colon adenocarcinoma (Dukes' stage B2) was minced, mixed with 0.5 ml of Matrigel (14 mg ml-') and injected subcutaneously into a nude mouse. The tumour from the third xenograft was enzymatically digested with trypsin-EDTA (0.05% trypsin in 0.53 mM EDTA) to yield a single-cell suspension. These cells were cultured in RPMI-1640 growth medium (Gibco-BRL) containing 10% fetal calf serum (Hyclone), insulin (5 jg ml-'), transferrin (5 ,ug ml-1), selenium (5 ug ml-') penicillin (100 g ml-'), streptomycin (100 jig ml-') and gentamicin (50 jig ml-')
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