NO38 expression and nucleolar counts are correlated with cellular DNA content but not with proliferation parameters...

To investigate the expression of nucleolar protein NO38, to determine the numbers of nucleoli per cell, and to examine the relations of these nucleolar parameters to tumour DNA index, total cellular DNA content, S phase fraction, and Ki67 labelling index.

36 colorectal tumours and 14 normal mucosas were studied. An anti-NO38 monoclonal antibody, 31A12, and flow cytometric analysis were used to detect expression of NO38 by means of a biotin-streptavidin-FITC (fluorescein isothiocyanate) staining method. Nucleolar counts were determined using fluorescence microscopy. Flow cytometry was used to determine tumour DNA indices and the sizes of the S phase fractions. Ki67 labelling indices were determined from tissue sections stained immunohistochemically with the MIB-1 antibody against the Ki67 nuclear protein.

Generally, tumour cell nucleoli were larger and more irregular in shape compared with nucleoli in normal mucosal cells. DNA aneuploid and diploid tumours expressed 2.8 and 2.1 times more NO38 than normal mucosa. The mean (SD) values for nucleolar counts were higher for the DNA aneuploid tumours (3.81 (0.93)) than the diploid tumours (2.62 (0.38)) and normal mucosa (2.34 (0.37)). NO38 expression and numbers of nucleoli correlated significantly (r = 0.52, p = 0.01). There were, however, no significant correlations between these nucleolar parameters and either the sizes of tumour S phase fractions or Ki67 labelling indices. Cell cycle resolved expression of NO38 in tumours and normal mucosa demonstrated that expression increased approximately in proportion to the DNA content throughout the cell cycle. In aneuploid tumours, NO38 expression was 43% and 98% higher in S and G2 phases, respectively, compared with the G1 phase. Sorting of these populations revealed that the nucleolar count also increased as the DNA content increased but by only 29% and 47% in S and G2, respectively. Apoptotic cells lacked NO38.

NO38 expression is higher in tumours than in normal mucosa owing to the increased DNA content and larger nucleoli in tumours; expression increases proportionally with DNA content as cells progress through the cell cycle from G1 through S and G2. However, NO38 expression does not correlate with the tumour S phase fraction or Ki67 labelling index and is lost during apoptosis. Also the results suggest that nucleoli grow in size during the cell cycle, which would account for the doubling of NO38 expression from G1 to G2, as the nucleolar count increased by only 47%.