A risk score model predictive of the presence of additional disease in the axilla in early-breast cancer patients with one or two metastatic sentinel lymph nodes.

BACKGROUND: Axillary lymph node dissection (ALND) in early-breast cancer patients with positive sentinel node (SLN+) may not always be necessary. AIMS: To predict the finding of ≥1 metastatic axillary node in addition to SLN+(s); to discriminate between patients who would or not benefit from ALND. METHODS: Records of 397 consecutive patients with 1-2 SLN+s receiving ALND were reviewed. Clinico-pathological features were used in univariate and multivariate analyses to develop a logistic regression model predictive of the risk of ≥1 additional axillary node involved. The discrimination power of the model was quantified by the area under the receiver operating characteristic curve (AUC) and validated using an independent set of 83 patients. RESULTS: In univariate analyses, the risk of ≥1 additional node involved was correlated with tumor size, grade, HER-2 and Ki-67 over-expression, number of SLN+s. All factors, but Ki-67, retained in multivariate regressions were used to generate a predictive model with good discriminating power on both the training and the validation sets (AUC 0.73 and 0.75, respectively). Three patient groups were defined based on their risk to present additional axillary burden. CONCLUSIONS: The model identifies SLN+-patients at low risk (≤15%) who could reasonably be spared ALND and those at high risk (>75%) who should receive ALND. For patients at intermediate risk, ALND appropriateness could be individually evaluated based on other clinico-pathological parameters.

Association of CTLA-4 polymorphisms with improved overall survival in melanoma patients treated with CTLA-4 blockade: a pilot study.

CTLA-4 blockade with monoclonal antibodies can lead to cancer regression in patients with metastatic melanoma (MM). CTLA-4 gene polymorphisms may influence the response to anti-CTLA-4 antibodies although few data are available regarding this issue. We analyzed six CTLA-4 single nucleotide polymorphisms (-1661A > G, -1577G > A, -658C > T, -319C > T, +49A > G, and CT60G > A) in 14 Italian MM patients and 45 healthy subjects. We found a significant association between the -1577G/A and CT60G/A genotypes and improved overall survival (Pc < 0.006, Bonferroni corrected), further confirmed by the diplotype analysis (-1577 & CT60 GG-AA diplotype, p < 0.001). A positive trend toward an association between these genotypes and response to therapy was also observed.

Accuracy of sentinel lymph node biopsy after neo-adjuvant chemotherapy in patients with locally advanced breast cancer and clinically positive axillary nodes.

BACKGROUND: Feasibility and accuracy of sentinel node biopsy (SLNB) after the delivery of neo-adjuvant chemotherapy (NAC) is controversial. We here report our experience in NAC-treated patients with locally advanced breast cancer and clinically positive axillary nodes, and compare it with the results from our previous randomized trial assessing SLNB in early-stage breast cancer patients. PATIENTS AND METHODS: Sixty-four consecutive patients with large infiltrating tumor and clinically positive axillary nodes received NAC and subsequent lymphatic mapping, SLNB and complete axillary lymph node dissection (ALND). The status of the sentinel lymph node (SLN) was compared to that of the axilla. RESULTS: At least one SLN was identified in 60 of the 64 patients (93.8%). Among those 60 patients, 37 (61.7%) had one or more positive SLN(s) and 23 (38.3%) did not. Two of the patients with negative SLN(s) presented metastases in other non-sentinel nodes. SLNB thus had a false-negative rate, a negative predictive value and an overall accuracy of 5.1%, 91.3% and 96.7%, respectively. All these values were similar to those we reported for SLNB in the settings of early-stage breast cancer. CONCLUSION: SLNB after NAC is safe and feasible in patients with locally advanced breast cancer and clinically positive nodes, and accurately predicts the status of the axilla.

Neo-adjuvant chemo/immunotherapy in the treatment of stage III (N2) non-small cell lung cancer: a phase I/II pilot study.

In a previous randomized study, we showed that adjuvant immunotherapy with tumor-infiltrating lymphocytes and recombinant interleukin-2 (rIL-2) significantly improved survival in resected N2-non small cell lung cancer (NSCLC) patients. The present study assesses feasibility, safety and potential efficacy of combined neo-adjuvant chemotherapy and immunotherapy with peripheral blood mononuclear cells (PBMC) and rIL-2 in resectable N2-NSCLC patients. Eighty-two consecutive N2-NSCLC patients underwent neo-adjuvant chemotherapy with cisplatin and gemcitabine. Out of the 82 patients, 23 were also subjected to leukapheresis prior to neo-adjuvant chemotherapy while the remaining 59 did not. Collected PBMC were analyzed for viability and phenotype and then stored frozen in liquid nitrogen. Thawed PBMC were infused intravenously, 5 days before surgery. After the infusion, rIL-2 was administered subcutaneously until surgery. Only patients with a partial or complete response to neoadjuvant chemotherapy underwent surgery: 13 patients in the experimental immunotherapy group (A) and 32 in the reference group (B). The two groups were homogeneous for all major prognostic factors. Median leukapheresis yield was 10 billion PBMC, (range 3-24 billions). Two to six billion PBMC were infused. The phenotypic analysis showed that similar proportions of CD4 and CD8 cells were present in leukapheresis products, and thawed PBMC, as well as in T lymphocytes isolated from the removed tumours. No severe adverse effects were observed following immunotherapy. No significant differences in overall survival (OS) and event-free survival (EFS) were seen between the two groups. However, the 5-year OS in group A was almost twice as much compared to group B (59 percent vs 32 percent). After adjustment for major prognostic factors, a statistically significant 66 percent reduction in the hazard of death was seen in patients receiving immunotherapy. The OS benefit was more evident in patients with adenocarcinoma than in those with squamous cell carcinoma. This study supports the favorable toxicity profile and potential efficacy of combining neo-adjuvant chemotherapy and immunotherapy with PBMC and rIL-2 in the treatment of N2-NSCLC patients.

Intra-operative evaluation of the sentinel lymph node for T1-N0 breast-cancer patients: always or never? A risk/benefit and cost/benefit analysis.

AIM: To investigate whether omitting intra-operative staging of the sentinel lymph node (SLN) in T1-N0 breast-cancer patients is feasible and convenient because it could allow a more efficient management of human and logistic resources without leading to an unacceptable increase in the rate of delayed axillary lymph node dissection (ALND). METHODS: According to the experimental procedure, T1a-T1b-patients were to not receive any intra-operative SLN evaluation on frozen sections (FS). In all T1c-patients, the SLN was macroscopically examined; if the node appeared clearly free of disease, no further intra-operative assessment was performed; if the node was clearly metastatic or presented a dubious aspect, the pathologist proceeded with analysis on FS. T2-patients, enrolled in the study as reference group, were treated according to the institutional standard procedure; they all received SLN staging on FS. RESULTS: The study included 395 T1-N0-patients. Among the 118 T1a-T1b-patients whose SLN was not analyzed at surgery, 12 (10.2%) were recalled for ALND. In the group of 258 T1c-patients, 112 received SLN analysis on FS and 146 did not. An SLN falsely negative either at macroscopic or FS examination was found in 33 (12.8%) cases. Overall, the rate of recall for ALND was 11.6% as compared to 8.4% in T2-patients. Using the experimental protocol, the institution reached a 9.6% cost saving, as compared to the standard procedure. CONCLUSIONS: Omission of SLN intra-operative staging in T1-N0-patients is rather safe. It provides the institution with both management and economical advantages.

Sentinel node biopsy compared with complete axillary dissection for staging early breast cancer with clinically negative lymph nodes: results of randomized trial.

BACKGROUND: Sentinel lymph node (SLN) staging is currently used to avoid complete axillary dissection in breast cancer patients with negative SLNs. Evidence of a similar efficacy, in terms of survival and regional control, of this strategy as compared with axillary resection is based on few clinical trials. In 1998, we started a randomized study comparing the two strategies, and we present here its results. MATERIALS AND METHODS: Patients were randomly assigned to sentinel lymph node biopsy (SLNB) and axillary dissection [axillary lymph node dissection (ALND arm)] or to SLNB plus axillary resection if SLNs contained metastases (SLNB arm). Main end points were overall survival (OS) and axillary recurrence. RESULTS: One hundred and fifteen patients were assigned to the ALND arm and 110 to the SLNB arm. A positive SLN was found in 27 patients in the ALND arm and in 31 in the SLNB arm. Overall accuracy of SLNB was 93.0%. Sensitivity and negative predictive values were 77.1% and 91.1%, respectively. At a median follow-up of 5.5 years, no axillary recurrence was observed in the SLNB arm. OS and event-free survival were not statistically different between the two arms. CONCLUSIONS: The SLNB procedure does not appear inferior to conventional ALND for the subset of patients here considered.

Timing of adjuvant chemotherapy and tamoxifen in women with breast cancer: findings from two consecutive trials of Gruppo Oncologico Nord-Ovest-Mammella Intergruppo (GONO-MIG) Group.

BACKGROUND: The timing of adjuvant chemotherapy and tamoxifen (TAM) has been investigated only in postmenopausal women with breast cancer. We analyzed the outcome of both pre- and postmenopausal women who entered two randomized trials (Gruppo Oncologico Nord-Ovest-Mammella Intergruppo studies) on adjuvant chemotherapy and received either concomitant or sequential TAM. PATIENTS AND METHODS: Patients who received anthracycline-based regimens and either concomitant or sequential TAM were eligible. The primary end point was overall survival (OS). Hazard ratios (HRs) of death or recurrence for treatment comparisons were estimated by Cox proportional hazards regression models. RESULTS: Among the 1096 eligible patients, 507 (46.3%) and 589 (53.7%) received concomitant and sequential TAM, respectively. The median follow-up time was 6.6 years. Ten-year OS was 83% [95% confidence interval (CI) 78-88%] and 80% (95% CI 74-86%) in the concomitant and sequential groups, respectively. Multivariate analyses confirmed no significant difference in the hazard of death (HR = 1.13; 95% CI 0.78-1.64; P = 0.534) and recurrence (HR = 1.03; 95% CI 0.80-1.33; P = 0.88) between the two groups. A decreasing trend (P = 0.015) in HR of death with increasing age was observed indicating, that concomitant therapy might be more effective than sequential therapy in young patients. CONCLUSIONS: We observed no outcome difference between sequential and concomitant chemo-endocrine therapy. The potential advantage of concomitant TAM in young patients needs to be further addressed in prospective trials.

Autologous chondrocyte implantation (ACI) for aged patients: development of the proper cell expansion conditions for possible therapeutic applications.

OBJECTIVE: Proliferation and chondrogenic commitment of cultured articular chondrocytes are impaired when cells derive from aged donors. In those subjects the feasibility of cell-based therapies for articular surface repair is reduced. Moreover, the use of serum as medium supplement elicits non-physiological responses in cultured chondrocytes. This study was therefore undertaken to identify the expansion culture conditions needed to sustain growth and chondrogenic commitment of chondrocytes harvested from aged human subjects. DESIGN: Articular cartilage was obtained from aged (69-75 years) and from young adult subjects (27-35 years). Chondrocytes were isolated and cultured in serum-free (SF) or in serum-supplemented [fetal calf serum (FCS)] conditions. Chondrocytes were expanded in monolayer for five duplications and processed for RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The differentiation potential was assessed by micromass pellet cultures before and after expansion in either culture medium, or after a prolonged exposure to serum followed by a period in SF condition. RESULTS: Only SF-cultured chondrocytes reached five duplications within 25-35 days, maintaining the expression of some chondrogenic markers and without altering the levels of active matrix metalloproteinase 3 (MMP-3). Only the pellets derived from SF-expanded cultures positively stained for cartilage matrix deposition. On the contrary, exposure to serum diminished the proliferation capacities, abolished the differentiation potential in the same cells and elicited transcription of the MMP-3 gene. Shifting culture conditions from FCS to SF resumed growth rates but proper extracellular matrix deposition was only partially restored. CONCLUSIONS: The SF conditions have proven valuable to prime cell proliferation and to sustain proper commitment in chondrocytes from aged patients. This culturing approach may represent a therapeutic chance extendable to a range of patients normally excluded from clinical protocols based on autologous chondrocyte implantation (ACI).

Extracellular fatty acid binding protein (ex-FABP) is a stress protein expressed during chondrocyte and myoblast differentiation.

OBJECTIVE: We have isolated and characterized in our laboratory a lipocalin specifically binding unsaturated long chain fatty acids (Ex-FABP). In developing chicken embryo long bones, Ex-FABP first appears at the boundary of the cone of hypertrophic cartilage. 'In vitro' EX-FABP is highly expressed by differentiating hypertrophic chondrocytes. Ex-FABP is expressed also in the forming myotubes both 'in vivo' and 'in vitro'. In cultured chondrocytes, Ex-FABP expression is strongly induced by treatment with inflammatory agents such as the bacterial liposaccharide LPS or interleukin-6. The possible mechanism for this induction was investigated. Expression of Ex-FABP was studied in other stress conditions. DESIGN: To investigate a possible mechanism for Ex-FABP induction by LPS or interleukin-6, we have cultured the cells in the presence of either hydrogen peroxide or the NO donor SNAP (S-nitrosil-acetil-D, L-penicillamine), two agents known to produce cellular stresses through the activation of specific signalling pathways. To investigate Ex-FABP expression in other stress conditions, chondrocytes were cultured for 3 days in the presence of alpha,alpha-dipyridyl, an agent inhibiting prolyl hydroxylase activity and collagen secretion. Supplement of this agent to the culture medium results in an impairment of collagen secretion and assembly and the consequent altered interaction of the cell with the surrounding extracellular matrix. In addition Ex-FABP expression was studied also in chondrocytes cultured in the absence of serum, a stress condition activating cell defence mechanisms. RESULTS: We have excluded that induction of Ex-FABP expression by inflammatory agents is mediated by oxidative stress or NO production. Ex-FABP expression was induced also by changes in the hypertrophic chondrocyte microenvironment, considered either as extracellular matrix surrounding the cell in culture or as nature and concentration of growth factor in the culture medium. CONCLUSIONS: No definitive data are so far available on the possible role of Ex-FABP when induced by cellular stresses. The capacity of the protein to specifically bind and transport unsaturated long chain fatty acids suggests that lipid metabolism and fatty acid utilization by the cells may be involved. Based on literature data the NRL/N-GAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) protein was proposed as a possible mammal counterpart of the chick Ex-FABP. We have suggested that Ex-FABP and NRL/NGAL expression in forming bones and muscles is part of a 'physiological' acute phase response. Interestingly the expression of Ex-FABP and NRL/NGAL is also activated in osteoarthritic cartilage and in the case of NRL/N-GAL during neoplastic transformation of chondrogenic lineage cells.

Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis.

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.

Developmental control of chondrogenesis and osteogenesis.

During vertebrate embryogenesis, bones of the vertebral column, pelvis, and upper and lower limbs, are formed on an initial cartilaginous model. This process, called endochondral ossification, is characterized by a precise series of events such as aggregation and differentiation of mesenchymal cells, and proliferation, hypertrophy and death of chondrocytes. Bone formation initiates in the collar surrounding the hypertrophic cartilage core that is eventually invaded by blood vessels and replaced by bone tissue and bone marrow. Over the last years we have extensively investigated cellular and molecular events leading to cartilage and bone formation. This has been partially accomplished by using a cell culture model developed in our laboratory. In several cases observations have been confirmed or directly made in the developing embryonic bone of normal and genetically modified chick and mouse embryos. In this article we will review our work in this field.

Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy.

Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion.

A divergent role of COOH-terminal domains in Nurr1 and Nur77 transactivation.

Orphan nuclear receptors such as Nurr1 and Nur77 have conserved amino acid sequences in the zinc finger DNA binding domains and similar COOH-terminal regions, but have no known ligands. These receptors can bind DNA sequences (response elements) as monomers and can also heterodimerize with the retinoid X receptor to activate transcription. We report here the identification and initial characterization of a novel COOH-terminal truncated isoform of Nurr1, Nurr1a. Internal splicing of Nurr1 generates a frameshift such that a stop codon is prematurely encoded resulting in a naturally occurring COOH-terminal truncation. Embryonic and postnatal mouse brain showed both Nurr1 and Nurr1a mRNAs expressed during development. To characterize essential COOH-terminal elements that may be deleted from Nurr1a and determine function in putative ligand binding, we created COOH-terminal deletion mutants. Nurr1, Nur77, and 3'-truncated mutants bind in gel mobility shift assays to the monomeric Nur77 response element (B1A-RE). However, in transient transfection assays, a truncation of as little as 15 Nurr1 COOH-terminal amino acids diminished transcriptional activation of B1A-thymidine kinase-chloramphenicol acetyltransferase reporter. This result was not seen for a similar Nur77 deletion mutant, Nur77-586. Unlike full-length Nurr1 and Nur77, transactivation by Nur77-586 was not augmented in response to the presence of retinoid-like receptor and 9-cis-retinoic acid. Thus, the interaction of putative ligand binding and transactivation for Nurr1 and Nur77 may function differently.

Modulation of commitment, proliferation, and differentiation of chondrogenic cells in defined culture medium.

The factors regulating the growth and development of mesenchymal precursor cells toward chondrogenesis are not well identified. We have developed a defined serum-free culture system that allows the proliferation of chick embryo chondrogenic cells and their maturation toward hypertrophic chondrocytes. Proliferation is obtained in adhesion in medium supplemented with insulin (Ins), Dexamethasone (Dex), and either basic fibroblast growth factor (FGF-2), platelet-derived growth factor bb, epithelial growth factor, or GH; the highest mitogenic response is induced by FGF-2 in synergy with Ins. Ins can be substituted by Ins-like growth factor I. When these cells are transferred into suspension culture in Ins/Dex and T3 without growth factor supplement, they undergo the complete chondrogenic development characterized by type X collagen synthesis and cellular hypertrophy. During differentiation, Ins cannot be substituted by Ins-like growth factor I. Chondrogenesis is also evidenced by the formation of hypertrophic cartilage when the medium is supplemented with ascorbic acid. If T3 is introduced in the proliferation phase, the cells fail to differentiate to hypertrophy in suspension unless bone morphogenetic protein-2 is added. Assays of ectopic tissue formation in nude mice, with cells implanted sc after adsorption on collagen sponge or porous hydroxyapatite ceramics, indicate that cells grown in Ins/FGF-2 reform mainly cartilage in vivo, whereas expansion in Ins/T3/Dex/FGF-2 leads to the formation of cartilage, bone, and adipose tissue.

Identification of a nuclear protein from rat developing brain as heterodimerization partner with thyroid hormone receptor-beta.

Thyroid hormone receptors (TR) are ligand-activated transcription factors that modulate the expression of certain target genes in a developmental and tissue-specific manner. These specificities are determined by the tissue distribution of the TR isoforms alpha1 and beta1, the structure of the thyroid hormone response element (TRE) bound by the receptor, and heterodimerization partners. Among these, retinoid X receptors (RXR) have been recognized as the principal partners for TR. The present work reports the identification of a novel nuclear protein from 19-day-old embryonic rat brain that displays a distinct interaction pattern with TR isoforms at the level of the TRE of two genes known to be differentially expressed and regulated by thyroid hormone (T3): the ubiquitous malic enzyme and the brain-specific myelin basic protein. Electrophoretic gel mobility shift assays demonstrate that only TRbeta1 forms a specific complex with the rat brain nuclear factor on the myelin basic protein-TRE, but not on the malic enzyme-TRE. Thus, the interaction is selectively determined by both the receptor isoform and the structure of the TRE. The expression of this brain nuclear factor is restricted to the perinatal period, when myelination is sensitive to T3. Gel supershift assays with RXR-specific antibodies indicate that this factor is not one of the known RXR isoforms. However, it is most likely a new member of the RXR subfamily because it could be supershifted with an antibody raised against the highly conserved DNA-binding domain of RXRs.

Computer-based technique for cell aggregation analysis and cell aggregation in in vitro chondrogenesis.

No quantitative methods are currently available to measure different aggregation parameters in cell cultures. In this paper we describe a computer-based technique for the automatic and reliable analysis of cellular aggregates, starting from optical microscopy images of living cells grown in suspension. The method allows determination, on the same sample at different time intervals, of quantitative parameters, including aggregation percentage, average number of cells in aggregates, and aggregate size statistical distribution. To determine the number of cells in an aggregate starting from its two-dimensional microscopic profile, a model has been proposed and verified, using sphere packing theory. Algorithms have been tested on chondrocyte suspension cultures, where cell aggregation is a very early and critical event leading to cell differentiation. Using this technique for the analysis of chick embryo chondrocyte cultures, we observed that aggregate size and development kinetics depend on the culture conditions used. The method, with minor adaptations, is of potential use also in other cell systems to evaluate aggregation indexes or to study aggregation kinetics.

Expression of the cartilage matrix protein gene at different chondrocyte developmental stages.

Cartilage matrix protein (CMP), a major noncollagenous component of certain types of hyaline cartilage, is synthesized by chondrocytes in a developmentally regulated manner. In this study, we monitored the accumulation of CMP in the developing chicken limb and sternum by immunostaining. In older embryos, the specific extracellular staining was restricted to the resting/proliferative zone of metaphyseal cartilage and to the immediately adjacent hypertrophic cartilage. A lack of staining was observed in the peripheral layers of articular cartilage. Data were compared with the accumulation of CMP mRNA measured by Northern analysis relative to other cartilage-specific messages in cell cultures representing different stages of chondrocyte differentiation, as well as with the steady state mRNA levels in tissue samples. We found a correlation between the gene expression pattern of the in vitro cultures and the one observed in certain in vivo differentiation stages. The high-density mesenchyme culture was utilized as a model for studying the events at early stage I (stage Ia) of chondrogenesis. This culture was characterized by relatively low steady state mRNA levels for cartilage proteins, including the later activation of the CMP gene as compared to type II collagen or link protein genes, and relatively high steady state mRNA levels for type VI collagen and beta-actin. Chicken embryo chondrocyte cultures obtained from sterna of 14-day-old embryos, however, consisted predominantly of stage Ib chondrocytes, and showed high steady state levels for cartilage proteins, but relatively lower levels for type VI collagen and beta-actin mRNAs. In accordance with the in vivo data, a relatively high steady state level was detected for CMP mRNA in cultures of hypertrophic (stage II) chondrocytes. We also performed transient expression assays in the various culture systems to study the role of the promoter upstream and intronic control regions in the tissue- and developmental stage-specific regulation of the CMP gene. We showed that the enhancer worked in a lineage-specific manner, by further stimulating the minimal promoter activity independent of the developmental stage of chondrocytes, while it did not in other tissues. The promoter upstream control regions, however, seemed to play a role in restricting the promoter activity to a certain chondrocyte developmental stage.

Type VI collagen expression is upregulated in the early events of chondrocyte differentiation.

Dedifferentiated chondrocytes cultured adherent to the substratum proliferate and synthesize large amounts of type I collagen but when transferred to suspension culture they decrease proliferation, resume the chondrogenic phenotype and the synthesis of type II collagen, and continue their maturation to hypertrophic chondrocyte (Castagnola et al., 1986, J. Cell Biol. 102, 2310-2317). In this report, we describe the developmentally regulated expression of type VI collagen in vitro in differentiating avian chondrocytes. Type VI collagen mRNA is barely detectable in dedifferentiated chondrocytes as long as the attachment to the substratum is maintained, but increases very rapidly upon passage of the cells into suspension culture reaching a peak after 48 hours and declining after 5-6 days of suspension culture. The first evidence of a rise in the mRNA steady-state levels is obtained already at 6 hours for the alpha 3(VI) chain. Immunoprecipitation of metabolically labeled cells with type VI collagen antibodies reveals that the early mRNA rise is paralleled by an increased secretion of type VI collagen in cell media. Induction of type VI collagen is not the consequence of trypsin treatment of dedifferentiated cells since exposure to the actin-disrupting drug cytochalasin or detachment of the cells by mechanical procedures has similar effects. In 13-day-old chicken embryo tibiae, where the full spectrum of the chondrogenic differentiation process is represented, expression of type VI collagen is restricted to the articular cartilage where chondrocytes developmental stage is comparable to stage I (high levels of type II collagen expression).(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis.

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.