Type X collagen in the human invertebral disc: an indication of repair or remodelling?

The short-chained type X collagen was once thought to be produced exclusively by hypertrophic chondrocytes during endochondral ossification. More recently, however, it has been found elsewhere, for example in articular cartilage. In the present study, the occurrence of type X collagen in the intervertebral disc has been investigated. Human disc tissues of varying pathologies were examined for the presence of type X collagen and expression of alpha1(X) mRNA by immunohistochemistry and in situ hybridization respectively. All samples of disc contained areas that were immunoreactive but to varying extents. In the disc itself, staining for the protein and alpha1(X) mRNA was seen frequently associated with cells of the nucleus pulposus, which were large and of hypertrophic appearance, most commonly found in degenerate discs, and also in areas of disorganized architecture, such as clefts. In addition, type X collagen, both protein and mRNA, was found in regions of the cartilage end-plate, which calcify ectopically in scoliotic patients. We suggest that type X collagen production may be a response of disc tissue cells to a stimulus, such as altered loading.

Distribution and configuration of c-myc RNA during transcriptional attenuation in differentiating cells in-situ.

Previous northern studies of c-myc RNA downregulation during early cellular differentiation have shown reduced levels of mature transcript within 6-24 h, attributed to attenuation of transcription at pause sites downstream of the P2 promoter. The transcription initiation rate has been shown to be decreased in some and increased in other such studies. We assessed the contribution of RNA trafficking to c-myc reduction during differentiation by examining the localisation and configuration of exon-specific transcripts, using oligonucleotide probes and fluorescent in-situ hybridisation, in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate. A 2,4-dinitrophenyl-labelled probe to c-myc exon 3 sequences gave a strong cytoplasmic and nucleolar hybridisation signal in undifferentiated cells, which decreased markedly after 24 h of differentiation. Nucleolar staining for c-myc RNA colocalised with that from a probe for ribosomal 28 S RNA. The signal from an exon 1 probe specific for sequences upstream of the c-myc P2 promoter was much weaker, but increased in the nuclei of differentiating cells, which possessed unusual ring-like or lamellar deposits, outside the nucleolus. These deposits appeared faintly together with nuclear staining with the exon 1 sense probe but not the exon 3 sense probe in differentiating cells. These findings demonstrate that within the first 24 h of differentiation, full-length c-myc RNA, which is compartmentalised as expected for a mature transcript, is considerably downregulated but nuclear primary RNA continues to be transcribed from exon 1. This is in a configuration similar to that reported for unspliced transcripts and is not elongated into exon 3. Antisense transcription with these RNA morphological features also occurs in exon 1 during differentiation. These results indicate significant changes in the intracellular dynamics of c-myc RNA during differentiation and support transcriptional attenuation and post-transcriptional processes, such as splicing, rather than reduced transcription initiation as the primary mechanism of c-myc downregulation in the early phases of differentiation.

Somatostatin receptor subtype mRNA expression in human colorectal cancer and normal colonic mucosae.

Somatostatin analogues may be useful novel agents in the systemic treatment of advanced colorectal cancer, as somatostatin inhibits proliferation in a wide variety of cell types. Here, we report the expression profiles of somatostatin receptor mRNAs in 32 pairs of malignant and normal colonic epithelia. Receptor subtype 2 (hSSTR2) mRNA was detected throughout nearly 90% of both malignant and normal tissue by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Subtype 5 (hSSTR5) mRNA was detected in 46% and 45% of tumour and mucosal samples respectively, but in 75% (9/12) of early-stage tumours (tubulovillous adenomas, Dukes' A and B) compared with 31% (5/16) of late-stage tumours (Dukes' C and 'D' tumours), 0.05>P>0.025 (chi2 with Yates' correction). There was also reduced expression of hSSTR5 in samples of metastatic tumour (11%, 1/9) compared with all tumour samples (56%, 18/32) 0.025>P>0.01 (chi2 with Yates' correction). Other hSSTRs (1, 3 and 4) were expressed infrequently. Thus, hSSTR2 expression is retained after malignant transformation in colonic epithelium and, although it may potentially be a target for antiproliferative therapy, its ubiquitous expression militates against this. hSSTR5 warrants investigation as a tumour suppressor.

Flow cytometric quantitation of sequence-specific mRNA in hemopoietic cell suspensions by primer-induced in situ (PRINS) fluorescent nucleotide labeling.

We modified a technique of RNA detection based upon primed in situ labeling of RNA (PRINS) to quantitate poly(A) and histone mRNA in HL60 cell suspensions by flow cytometry. The direct PRINS technique was used to label poly(A) mRNA, based upon the incorporation of fluoresceinated nucleotides into cDNA chains generated by reverse transcription from the site of oligo(dT) primer-specific annealing on the poly(A) template. By this method, poly(A) fluorescent signals of up to 20-fold above background were obtained when primer concentrations were saturating. Nonspecific binding of the fluorochrome-conjugated nucleotide proved to be the greatest source of background fluorescence in cell suspensions. This was considerably reduced by preincubation of the cells in lysine and an excess of cold nucleotides prior to the PRINS reaction. The application of indirectly fluoresceinated antibodies to the FITC moiety of the conjugated nucleotide (indirect PRINS) enhanced detection of a fluorescent signal generated from cells by the direct PRINS technique after stringent histone priming. Cell cycle analysis, based upon DNA histograms obtained after PRINS labeling, showed the histone fluorescence to be primarily in S-phase cells of an exponentially growing population. Confocal laser scanning microscopy revealed good retention of cellular architecture, with cytoplasmic localization of poly(A) and histone mRNA fluorescence and heterogeneous mRNA expression among cells. Our modified PRINS labeling of RNA represents a useful advance in the precise quantitation of RNA species in cell suspension by flow cytometry.

c-myc and c-myb oncoproteins during induced maturation of myeloid and erythroid human leukemic cell lines.

c-myc and c-myb mRNAs have been found to be tightly regulated during hemopoietic differentiation. We have studied nuclear c-myc and c-myb oncoproteins through the cell cycle, during macrophage, granulocyte, erythroid, and megakaryocytic differentiation of KG1, HL60, and HEL cells. p62c-myc and p75c-myb content of propidium iodide-stained nuclei was quantitated by flow cytometry using fluoresceinated antibodies CT14-G4 and MB4.3, respectively. In uninduced cells p62c-myc content is highest in HL60, followed by HEL, then KG1, while p75c-myb is highest in HEL, followed by HL60 and KG1. All lines showed a less than 2-fold increment in both oncoproteins over the cell cycle. Macrophage induction of KG1 and HL60 resulted in early increase in both oncoproteins, followed by a decline to less than starting values by 48 h, concurrent with a reduction of S phase cells and the appearance of adherent alpha-naphthyl acetate esterase-positive cells. p62c-myc changes were more pronounced in HL60 and p75c-myb changes in KG1. Different patterns of oncoprotein expression were found when different inducing agents were used for granulocyte differentiation of HL60. Under all conditions, however, both oncoproteins declined to basal levels before granulocyte maturity. Hemin-induced erythroid differentiation of HEL to hemoglobin-containing cells resulted in biphasic p62c-myc and p75c-myb kinetics. In contrast, dimethyl sulfoxide-induced megakaryocytic differentiation of HEL was accompanied by an early and steady decline in both oncoproteins. Despite considerable reduction in oncoprotein levels, HEL cells were still actively cycling at 120 h. It appears that c-myc and c-myb proteins decline with differentiation, well before proliferation ceases in some lineages. The kinetics of the decline differ between the two oncogenes and vary with the lineage induced and the nature of the inducing agent used. The cell cycle distribution of the oncoproteins does not change during maturation. These data suggest disparate roles for c-myc versus c-myb during hemopoietic differentiation and the existence of multiple signal transduction pathways for down-regulation of these genes.

c-myc protein kinetics during growth and differentiation of CD34 (MY10)-positive blast cells from normal human marrow.

We have used flow cytometry to quantitate nuclear c-myc protein, at each phase of the cell cycle, during in-vitro differentiation of CD34-positive stem cells isolated from normal human bone marrow by the monoclonal antibody, MY10. Mean c-myc protein levels in CD34-positive cells, consisting of greater than 70% blasts, are lower than a marrow fraction containing myeloid cells of intermediate maturation, but have an invariant proportional relationship, with regard to nuclear mass, over the cell cycle. The majority of these primitive cells are non-cycling, as revealed by DNA content. Under our assay conditions, nuclear c-myc protein distribution over the cell cycle did not change as these progenitors entered a proliferative phase in culture. In cultures containing factors supporting myeloid maturation, mean G0/G1 p62c-myc levels initially decline, then rise above starting values as promyelocytes and myelocytes differentiate from CD34-positive cells, and as proliferation begins. With further myeloid maturation, and while cell numbers are increasing, c-myc protein continues to increase. C-myc protein kinetics differ in cultures in which macrophages, rather than myeloid cells, predominate. These data indicate that a complex relationship exists between c-myc gene expression and proliferation, maturation and lineage in haemopoietic cells, and lend support to the notion that early down regulation may be causally associated with the differentiation process.

Nuclear c-myc protein, maturation, and cell-cycle status of human haemopoietic cells.

Human c-myc protein, p62c-myc, has been quantitated by flow cytometry in the nuclei of normal marrow and peripheral blood cells, and the HL60 cell line. Marrow and peripheral blood cells exhibit nuclear c-myc protein throughout the cell-cycle, at an average level 2-3-fold lower than HL60 cells. In no cells did p62c-myc vary more than 2-fold throughout the cell cycle. A small subset of marrow G0/G1 cells, enriched in early myeloid and blast cell fractions, contained p62c-myc at levels equal to or even exceeding those of HL60. Overall c-myc protein content was higher in myeloid, compared to erythroid and lymphoid marrow fractions. Within the myeloid lineage, the highest average p62c-myc level was present in cells of intermediate maturation, i.e. myelocytes and metamyelocytes. In the erythroid lineage, c-myc protein level was highest in the most immature cells and declined with maturation. Significant amounts of p62c-myc were present in post-mitotic, end-stage neutrophils, but were barely detectable in cycling late erythroblasts or in quiescent lymphocytes and monocytes. HL60 cells, despite c-myc gene amplification and increased gene expression, contain c-myc protein at a level corresponding to promyelocytes in normal human marrow. The virtual absence of p62c-myc in cycling late erythroblasts, and its presence in post-mitotic end-stage granulocytes, suggests that c-myc protein may have functions unrelated to cell proliferation.

Regulation of lymphoid colony growth by factors derived from human bone marrow.

We have previously demonstrated the ability of soluble factors derived from cultured murine and human bone marrow supernatants to modulate a variety of lymphoid functions, including DNA synthesis. In the present report, we show that human marrow supernatants contain a suppressor factor (BSF) that suppresses T-lymphoid colony formation, and an augmenting factor (BEF) that enhances T-colony growth. BSF suppression exhibits no tissue specificity, affecting marrow-derived and peripheral T colonies similarly. The suppressive activity occurs prior to mitogenic activation by TCGF. In contrast, a preferential augmentation of the size and number of marrow-derived T-cell colonies, as compared to peripheral T-cell colonies, was observed in the presence of BEF. BEF required prior mitogen activation of the colony inocula to effect colony enhancement. In addition, the response to BEF was greater for E- than for E+ colony-forming cells, indicating the target of BEF activity to be an early T cell. The active subfraction of BEF with colony-enhancing activity was found to be between 8000 and 30,000 daltons.

Immunoregulatory activity of human bone marrow. Identification of suppressor cells possessing OKM1, SSEA-1, and HNK-1 antigens.

Human bone marrow contains natural regulatory cells capable of suppressing the in vitro primary IgM response of normal tonsillar cells. The suppression is mediated by non-T cells possessing Fc receptors, OKM1, SSEA-1, and HNK-1 antigens on their surface. The suppression was abrogated by treatment of bone marrow cells (BMC) with anti-HNK-1 or anti-SSEA-1 antisera and complement. Furthermore, BMC depleted of HNK-1+ cells could respond in a primary in vitro antibody response when provided with accessory T cells and macrophages from tonsillar cells. Our findings support the idea that HNK-1+ and HNK-1- BMC populations act antagonistically in the regulation of antibody synthesis. Further, the finding of HNK-1+, SSEA-1+, and OKM1+ suppressor cells in human bone marrow may represent a precursor phenotype of mature natural killer cells with potent immunoregulatory activity.