Expression of type X collagen and matrix calcification in three-dimensional cultures of immortalized temperature-sensitive chondrocytes derived from adult human articular cartilage.

Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.

Growth factor control of rat thyroid follicular cell proliferation.

We have investigated the proliferative responses of rat thyroid follicular cells in serum-free culture to a range of growth factors including TSH, epidermal growth factor, and insulin, added singly or in combination. Follicles released from normal thyroids by collagenase/dispase digestion were cultured in suspension in agarose-coated microtiter plates to prevent monolayer formation. Growth responses were measured by [3H] thymidine incorporation and by autoradiography over successive 24- or 36-h periods. Insulin, even in the absence of other growth factors, stimulated [3H]thymidine incorporation in a concentration-dependent manner, rising from basal levels of 486 +/- (SE) 18 cpm to 4222 +/- 367 cpm/5 X 10(4) cells at 8 micrograms/ml. In contrast, TSH alone had no effect. In the presence of threshold levels (0.08 micrograms/ml) of insulin, however, there was a highly significant (P less than 0.001) response to TSH, [3H]thymidine incorporation rising from 1089 +/- 163 cpm in the absence of TSH to a maximum of 7548 +/- 585 with 1 mU/ml TSH. There was a synergistic interaction between insulin and TSH over the concentrations tested. Epidermal growth factor either alone or in combination with insulin failed to produce a significant response. Parallel autoradiographic studies were concordant with the [3H]thymidine incorporation data. We conclude that whereas in the absence of other growth factors TSH is unable to stimulate DNA synthesis in isolated rat thyroid follicles, the inclusion of just a single growth factor, insulin, permits a marked response. These observations emphasize the need for inclusion of appropriate permissive growth factor(s) when assessing the in vitro effect of a suspected tissue-specific mitogen.

In vitro evidence for an intracellular mechanism limiting the thyroid follicular cell growth response to thyrotropin.

The purpose of this study was to further investigate the mechanism that limits thyroid growth in the presence of a sustained elevation of serum TSH. An in vitro thyroid follicle culture was used, thyroid function and growth being assessed by 131I- organification and [3H]thymidine incorporation into DNA, respectively. Normal thyroid follicles incorporated [3H]thymidine in response to added TSH and also organified 131I-. Follicles taken from rats previously given goitrogen for 80 days, however, organified 131I- in response to added TSH but did not incorporate [3H]thymidine. These in vitro results parallel those obtained in in vivo studies despite the disruption of thyroid architecture. We conclude that the growth desensitization seen in vivo during sustained serum TSH elevation is mediated by an intracellular change in the follicular cell (either at the receptor or postreceptor level) rather than by a locally acting chalone.

The effects of hyperthyroidism on experimental autoimmune thyroiditis in the rat.

The effects of hyperthyroidism on experimental autoimmune thyroiditis were investigated in the rat. Rats were given T4 twice daily by sc injection in amounts sufficient to raise circulating hormone levels 10-fold 4 h after administration. Thyroiditis was induced by immunization with rat thyroglobulin (Tg) in complete Freund's adjuvant, and the severity of the disease was assessed by comparison with saline-treated controls. Thymic and splenic hypertrophy were found in T4-treated animals, whereas lymph node wt decreased. The levels of Tg antibodies did not differ between animals given saline and those given T4, but the expected sustained rise in control animals was not seen in those treated with T4; in addition, there was a significant decrease in the amount of Tg antibody produced by in vitro culture of lymph node lymphocytes from T4-treated rats. Continuous T4 administration lowered the number of T cells in the circulation, but the number of phenotypically identified helper cells remained the same. The most striking effects of T4 were to ameliorate the intensity of histologically defined thyroiditis and lower the response of lymph node T cells to the nonspecific mitogen, phytohemagglutinin. These results show that excessive T4 does not, as previously suggested, enhance the immune response in autoimmune thyroid disease: on the contrary, suppression is found with the dose and model we have used. In view of the magnitude of this effect, it is now important to identify the site of T4 action and investigate how this effect contributes to the autoimmune response in Graves' disease.

Genetic regulatory elements introduced into neural stem and progenitor cell populations.

The genetic manipulation of neural cells has advantage in both basic biology and medicine. Its utility has provided a clearer understanding of how the survival, connectivity, and chemical phenotype of neurones is regulated during, and after, embryogenesis. Much of this achievement has come from the recent generation by genetic means of reproducible and representative supplies of precursor cells which can then be analyzed in a variety of paradigms. Furthermore, advances made in the clinical use of transplantation for neurodegenerative disease have created a demand for an abundant, efficacious and safe supply of neural cells for grafting. This review describes how genetic methods, in juxtaposition to epigenetic means, have been used advantageously to achieve this goal. In particular, we detail how gene transfer techniques have been developed to enable cell immortalization, manipulation of cell differentiation and commitment, and the controlled selection of cells for purification or safety purposes. In addition, it is now also possible to genetically modify antigen presentation on cell surfaces. Finally, there is detailed the transfer of therapeutic products to discrete parts of the central nervous system (CNS), using neural cells as elegant and sophisticated delivery vehicles. In conclusion, once the epigenetic and genetic controls over neural cell production, differentiation and death have been more fully determined, providing a mixture of hard-wired elements and more flexibly expressed characteristics becomes feasible. Optimization of the contributions and interactions of these two controlling systems should lead to improved cell supplies for neurotransplantation.

Immortalization of human marrow stromal cells by retroviral transduction with a temperature sensitive oncogene: identification of bipotential precursor cells capable of directed differentiation to either an osteoblast or adipocyte phenotype.

The etiology of osteoporosis is multifactorial, but there is evidence from both animal and human studies that the volume of marrow adipose tissue increases when bone volume is reduced in osteoporosis. The cell-related mechanism that may account for this inverse relationship between the volume of marrow adipose tissue and bone remains to be clarified, although it is known that both adipocytes and osteoblasts are derived from stromal cells precursors in bone marrow. We report that retroviral transduction with a temperature-sensitive oncogene (SV40 large T antigen) can generate bipotential cell lines from human marrow stroma that are capable of directed differentiation, in vitro, down either an osteogenic or adipocytic lineage pathway. One such clone, designated hOP 7, expresses type alpha 1(I) procollagen and has low alkaline phosphatase (AP) activity under basal culture conditions that is reminiscent of an osteoprogenitor cell. Exposure of hOP 7 cells to dexamethasone upregulates AP activity and enables the cells to mineralize their extracellular matrix. Also, treatment with calcitriol induces osteocalcin expression and both PTH and PGE2 induce/augment cAMP formation. Incubation with normal rabbit serum, however, causes the cells to become adipogenic as demonstrated by histological staining with Oil-red-O, expression of mRNA for the early and late adipocyte markers lipoprotein lipase and glycerol 3-phosphate dehydrogenase, respectively, and loss of type alpha 1(I) procollagen mRNA. The generation of homogeneous populations of these cells, as confirmed by Southern blot analysis, demonstrates the capacity of a human clonal cell line to differentiate in either an osteogenic or adipogenic direction.

Identification of 5-HT receptor sub-types in a homogeneous population of presumptive serotoninergic neurones.

The expression of the known rat 5-HT receptor sub-types has been analyzed in a presumptive serotoninergic cell line derived from the rat raphé nuclei, using reverse transcription followed by polymerase chain reaction. By manipulating the activity of the oncogene (ts-SV40T) product used to immortalize the serotoninergic precursors, it has been possible to compare the expression of the 5-HT receptors in either replicative or differentiating cells. 5-HT1B, 5-HT1D, 5-HT3, 5-HT6 and 5-HT7 receptor gene expression were all observed in the replicating cells. However, under differentiation conditions, expression of all except the 5-HT1B receptor was lost. Only one novel amplification product appeared during early differentiation, in the 5-HT2B lane; its smaller than expected size was suggestive of a previously undescribed alternate splicing of the mRNA in brain. The curtailment of 5-HT receptor expression in differentiating neurones in vitro may reflect the normal ongoing restriction in the phenotypic potential during embryogenesis in vivo. The serotonin cells, therefore, constitute a pristine cell line in which to study the receptor pharmacology of one or more 5-HT receptor sub-types in isolation.

Ionic mechanisms underlying excitatory effects of serotonin on embryonic rat motoneurons in long-term culture.

The actions of serotonin were investigated on motoneurons isolated from embryonic day 14 rat spinal cord and enriched by metrizamide density gradient centrifugation. Trophic support was provided by a spinal cord glial monolayer, ciliary neurotrophic factor and heat-inactivated serum. Cultures were maintained for 17-83 days and investigated using whole-cell patch-clamp recording. Serotonin evoked slow depolarizations (6.2+/-0.7 or 9.3+/-1.3 mV in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione and strychnine, EC50 8.2 nM), which were reversibly blocked by 0.1 microM ketanserin. Serotonin generated synaptic potentials in motoneurons, lowered the threshold for repetitive firing and changed the slope of the current intensity-firing frequency relationship. The inward current evoked by serotonin (-147+/-15.2 pA) was ascribed to a complex ionic mechanism, which varied amongst neurons in the sampled population. It was due to closure of barium-sensitive potassium channels, effects on Ih and increase in a separate mixed cation current which comprised both transient voltage-sensitive and sustained components. We conclude that serotonergic responses develop in motoneurons cultured under these conditions in the absence of serotonergic input, sensory neurons or many interneurons.

Abnormal expression of the MOS proto-oncogene in human thyroid medullary carcinoma.

We have been studying the expression of a range of proto-oncogenes in human thyroid tumour tissue by using Northern blot analysis. We have demonstrated the expression of a MOS mRNA of 1 kb in all thyroid samples. Furthermore, in a medullary carcinoma sample we also observed additional mRNA species of 1.7 and 2.2 kb. Southern blot analysis of DNA prepared from the same tumour sample did not reveal a rearrangement of the gene. These findings are the first report of MOS expression in any human tissue, and indicate that MOS oncogene activation might be important in the development of some thyroid tumours.

p53 protein expression in malignant, pre-malignant and non-malignant lesions of the lip.

AIM: To elucidate the role of the p53 tumour suppressor gene in the pathogenesis of lip cancer. METHODS: Expression of p53 was evaluated immunocytochemically in a retrospective study of formalin fixed, paraffin wax embedded tissue. Five cases each of four types of lip lesions were studied; these comprised squamous cell carcinoma (SCC), solar keratosis (SK), chronic hyperplastic candidosis (CHC), and lichen planus (LP). Five cases each of normal lip mucosa, SCC, and SK from sun exposed facial skin as well as LP, CHC, and SCC from buccal mucosa were also analysed. Immunolocalisation of p53 was scored semiquantitatively. The degree of apoptosis was also assessed in selected lesions by determining cell nuclear fragmentation. RESULTS: All SCCs from lip lesions were immunopositive for p53. All cases of SK and two of five CHC lip lesions were also p53 positive. Normal lip mucosa samples were p53 negative. Sun exposed skin lesions of SCC and SK were all positive for p53, but only three of five cases of SCC from the buccal mucosa had detectable levels of p53. p53 expression was not detected in CHC and LP lesions of the buccal mucosa. CONCLUSIONS: The aberrant expression of p53 is likely to occur early in the pathogenesis of lip cancer and may be related to exposure to the sun. The immunopositive p53 cells identified in the benign LP lesions do not necessarily correlate with commitment of cells within the lesion to programmed cell death. In light of the prior reports which indicate that p53 positive cells may progress to form malignant tumours, it is suggested that patients with p53 positive but otherwise benign lesions should be followed more closely.

Raphé neural cells immortalized with a temperature-sensitive oncogene: differentiation under basal conditions down an APUD cell lineage.

Dividing cells from the midline of the ventral rhombencephalon and medulla oblongata have been transduced with a modulatable oncogene, (ts)SV40-T, using retroviral gene transfer. At the permissive temperature of the oncogene (33 degrees C), cells replicated and were isolated as individual, homogeneous clones. The effects of simply raising the temperature to the oncogene's non-permissive value, namely 39 degrees C, were analyzed by immunohistochemical methods. In one clone in particular (921202-6), cells ceased replication and started to differentiate. Certain neuronal characteristics became apparent: neurone-specific enolase-like immunoreactivity developed, as did the ability to take up exogenously applied 5-hydroxytryptamine (5HT). In addition, the cells took up exogenous 5-hydroxytryptophan (5HTP), and subsequently decarboxylated it to 5HT. However, they were unable to synthesize immunohistochemically detectable amounts of 5HT using L-tryptophan as a precursor. No 5HT uptake was found either in mitotic cells of this clone held at 33 degrees C, or in several other neuronal clones differentiating at 39 degrees C. Neither the neuronal nor the serotoninergic characteristics of clone 921202-6 developed in the presence of retinoic acid. It is concluded that 921202-6 cells differentiate under basal conditions down a neuronal pathway typical of an APUD cell, and that the choice of this pathway is made prior to the end of cell cycling. Furthermore, predisposition of the precursor cells to the neuronal/APUD phenotype can be overridden by extraneous epigenetic factors.

Detection of a novel mutation in X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous group of inherited disorders of defective enamel formation. The major protein involved in enamel formation, amelogenin, is encoded by a gene located at Xp22.1-Xp22.3. This study investigated the molecular defect producing a combined phenotype of hypoplasia and hypomineralization in a family with the clinical features and inheritance pattern of X-linked amelogenesis imperfecta (XAI). Genomic DNA was prepared from buccal cells sampled from family members. The DNA was subjected to the polymerase chain-reaction (PCR) in the presence of a series of oligonucleotide primers designed to amplify all 7 exons of the amelogenin gene. Cloning and sequencing of the purified amplification products identified a cytosine deletion in exon VI at codon 119. The deletion resulted in a frameshift mutation, introducing a premature stop signal at codon 126, producing a truncated protein lacking the terminal 18 amino acids. Identifying mutations assists our understanding of the important functional domains within the gene, and finding another novel mutation emphasizes the need for family-specific diagnosis of amelogenesis imperfecta.

Importance of maintaining species homology in thyroid hormone radioimmunoassays: modification of 'human' radioimmunoassay kits for use with rat samples.

Lack of species homology in radioimmunoassays can lead to serious errors. We have shown that a highly significant bias results from application of commercially available 'human' thyroxine and triiodothyronine assays to rat samples. This paper describes a simple modification whereby this source of error is overcome. Validation of the modified assays demonstrated their high degree of reliability.

Mast cell hyperplasia during goitrogen-induced thyroid growth - a quantitative study.

This paper describes the effect of long-term goitrogen-administration on the mast cell population of the rat thyroid. Animals were treated with the goitrogen aminotriazole at a dose sufficient to block thyroid hormone synthesis completely, and compared after 80 days with a control group. Serum TSH was measured by radioimmunoassay, and mast cell number quantified from specially-stained tissue sections using appropriate serological methods. Goitrogen treatment led to a 9-fold increase in serum TSH and an 8-fold increase in thyroid weight. Mast cell number per unit volume of thyroid decreased, but total numbers per gland increased 4 to 5 fold. There was a significant fall in mean mast cell size. The work demonstrates conclusively that mast cell hyperplasia occurs during goitre formation and provides a method for its quantitative assessment. The possible mechanisms and significance of thyroid mast cell proliferation are discussed.

Circadian rhythm of mitotic activity in normal and hyperplastic rat thyroid.

This study was designed to provide a reliable quantitative assessment of the circadian rhythm of mitotic activity in the follicular cell population of the rat thyroid, and the effect on this rhythm of prolonged stimulation by a raised level of circulating TSH, induced by goitrogen administration. Mitotic activity in groups of control and goitrogen-treated rats was assessed by a stathmokinetic technique during four 4-h periods spaced equally through one 24-h cycle. Particular attention was paid to the method of sampling to eliminate systematic and minimise random errors, and to the assessment of rhythmicity which was carried out by an appropriate statistical method. A highly significant circadian rhythm was found in control animals with a daytime peak (12.00 to 16.00 h). Goitrogen treatment led to a 5- to 6-fold increase in the mean but a loss of detectable rhythmicity. The results show that the presence and timing of this circadian rhythm must be taken into account in future studied of thyroid growth, and they throw some light on the possible mechanisms of its control. Comparison of the rhythm with that of serum TSH reported previously raises the possibility of a dominant control by this hormone even in euthyroid animals and suggests that it may act on cells in the G2 and/or G1/G0 phases of the cell cycle.

Dissociation of growth and function in the rat thyroid during prolonged goitrogen administration.

This study was designed to investigate the changes in growth and function which occur in the rat thyroid during prolonged TSH stimulation. Animals maintained on the goitrogen aminotriazole were sacrificed together with controls at frequent intervals over a period of 5 months. The levels of serum T3 and T4 and TSH were measured by radioimmunoassay. Functional activity was assessed by measurement of the thyroid/serum iodide ratio (T/S) and growth by measurement of thyroid weight, follicular cell number and follicular cell mitotic activity. Serum T3 and T4 rapidly fell to undetectable levels within 2 weeks. The level of serum TSH rose to a stable 5-fold maximum after 4 weeks. The T/S ratio followed a closely similar pattern rising to a sustained 7-fold maximum. Thyroid weight and follicular cell number increased rapidly for the first few weeks but the growth rate declined progressively, falling almost to zero after 80 days. Mitotic activity rose dramatically to a 30-fold peak after 7 days but then declined almost to normal after 80 days, consistent with the observed change in cell number. The results thus demonstrate a clear dissociation between the functional and proliferative activity of the thyroid follicular cells during prolonged stimulation by a sustained elevation of serum TSH and point to the existence of specific growth regulating mechanisms which limit the mitotic response.

Physical randomization of tissue architecture: an alternative to systemic sampling.

A method is described for the rapid and accurate quantitation of tissue morphology using rat thyroid as a model. Perfused-fixed rat thyroid was diced into approximately thirty small pieces which were then randomly embedded in epon. Sections were taken at 500 micrometer intervals and stained with toluidine blue. Epithelial, follicular lumen and stromal components were quantified by a point counting technique. An analysis of variance was then performed on the data to determine whether there was any significant variation in the distribution of components between sections cut at different levels. No significant variation in any component was seen between sections for normal diced thyroid. This was also the case for diced thyroids of animals chronically treated with goitrogen to create a different physiological state. The method is therefore reliable even when marked changes in component distribution are induced. The physical randomization of tissue architecture by dicing, prior to embedding, greatly reduces the number of sections needed for the accurate morphometry of non-randomly distributed tissues.

Effect of raised serum prolactin on breast development.

The effect of serum prolactin elevation on the growth and development of the rat breast was investigated. Oral administration of the dopamine antagonist, perphenazine, led to a 5-10-fold elevation of serum prolactin after two days of treatment which was maintained for the 54 days of study. A significant (P less than 0.01) 3.4-fold increase in total breast volume was seen by Day 4 of serum prolactin elevation. Breast volume continued to rise up to Day 14 reaching an 8.9-fold peak (P less than 0.001) which was maintained for the duration of the experiment. Epithelial, myoepithelial, lumen and stromal volume changes in the ductular and alveolar compartments were quantified separately. Highly significant (P less than 0.01) volume increases were seen in all components within the first few days of prolactin elevation. Similar time courses of the growth response to elevated serum prolactin were seen in the ductal tissues reaching an approximate 3-fold peak by 7 days in duct epithelium, myoepithelium and duct stroma. Time coordinated growth responses were also seen in the alveolar tissues with larger (7-15-fold) increases in alveolar epithelium, alveolar myoepithelium and alveolar stroma, reaching a peak by 14 days.

Reparative growth in the rat thyroid: effect of TSH suppression.

Previous investigations have shown that thyroid incision leads to a dramatic burst of follicular cell mitotic activity in cells adjacent to the wound edge in both normal rats and rats made hypothyroid by chronic goitrogen administration. Wound-induced thyroid mitotic activity therefore, is seen in rats with either normal or supranormal levels of circulating thyrotropin (TSH). This study was designed to investigate the thyroid mitotic response to wounding in the absence of detectable levels of circulating TSH. Rats were injected with large doses of L-thyroxine twice daily to render circulating TSH undetectable. Thyroids were incised and follicular cell mitotic activity, in relation to distance from the incision, determined at 24, 48 and 72 hr after incision. A mitotic response to wounding was maintained in L-thyroxine treated rats, even though circulating TSH was undetectable. The peak of activity was at 48 hr, but was only 50% of that found in the incised normal rat thyroid. The spatial distribution of the response suggests that there are two components of the wound response in the normal thyroid, one dependent on the presence of circulating TSH, the other TSH-independent. The results are discussed in relation to current understanding of cellular growth control.