Melanoma secretion of transforming growth factor-ß2 leads to loss of epidermal AMBRA1 threatening epidermal integrity and facilitating tumour ulceration.

BACKGROUND: For patients with early American Joint Committee on Cancer (AJCC)-stage melanoma the combined loss of the autophagy regulatory protein AMBRA1 and the terminal differentiation marker loricrin in the peritumoral epidermis is associated with a significantly increased risk of metastasis. OBJECTIVES: The aim of the present study was to evaluate the potential contribution of melanoma paracrine transforming growth factor (TGF)-ß signalling to the loss of AMBRA1 in the epidermis overlying the primary tumour and disruption of epidermal integrity. METHODS: Immunohistochemistry was used to analyse AMBRA1 and TGF-ß2 in a cohort of 109 AJCC all-stage melanomas, and TGF-ß2 and claudin-1 in a cohort of 30 or 42 AJCC stage I melanomas, respectively, with known AMBRA1 and loricrin (AMLo) expression. Evidence of pre-ulceration was analysed in a cohort of 42 melanomas, with TGF-ß2 signalling evaluated in primary keratinocytes. RESULTS: Increased tumoral TGF-ß2 was significantly associated with loss of peritumoral AMBRA1 (P < 0·05), ulceration (P < 0·001), AMLo high-risk status (P < 0·05) and metastasis (P < 0·01). TGF-ß2 treatment of keratinocytes resulted in downregulation of AMBRA1, loricrin and claudin-1, while knockdown of AMBRA1 was associated with decreased expression of claudin-1 and increased proliferation of keratinocytes (P < 0·05). Importantly, we show loss of AMBRA1 in the peritumoral epidermis was associated with decreased claudin-1 expression (P < 0·05), parakeratosis (P < 0·01) and cleft formation in the dermoepidermal junction (P < 0·05). CONCLUSIONS: Collectively, these data suggest a paracrine mechanism whereby TGF-ß2 causes loss of AMBRA1 overlying high-risk AJCC early-stage melanomas and reduced epidermal integrity, thereby facilitating erosion of the epidermis and tumour ulceration.

Potentiating cutaneous wound healing in young and aged skin with nutraceutical collagen peptides.

BACKGROUND: Chronic wounds continue to be a burden to healthcare systems, with ageing linked to increased prevalence of chronic wound development. Nutraceutical collagen peptides have been shown to reduce signs of skin ageing, but their therapeutic potential for cutaneous wound healing remains undefined. AIM: To determine the potential for nutraceutical collagen peptides to promote cutaneous wound healing in vitro in the context of age. METHODS: The potential for bovine- or porcine-derived nutraceutical collagen peptides to promote wound healing of primary cutaneous fibroblasts and keratinocytes derived from young and aged individuals in vitro was assessed by two-dimensional scratch and cell-viability assays and by immunofluorescence for the cell proliferation marker, Ki67. The achievement of peptide concentrations in vivo, equivalent to those exerting a beneficial effect on wound healing in vitro, was confirmed by pharmacokinetic studies of hydroxyproline, a biomarker for collagen peptide absorption, following peptide ingestion by healthy individuals over a wide age range. RESULTS: Results demonstrated significant nutraceutical collagen peptide-induced wound closure of both young and aged fibroblasts and keratinocytes, mediated by enhanced cellular proliferation and migration. Analysis of blood levels of hydroxyproline in young and aged individuals following porcine collagen peptide ingestion revealed peak serum/plasma levels after 2 h at similar concentrations to those exerting beneficial effects on wound healing in vitro. CONCLUSION: These data demonstrate the capacity for nutraceutical collagen peptides to promote cutaneous wound closure in vitro, at pharmacologically achievable concentrations in vivo, thereby offering a potential novel therapeutic strategy for the management of cutaneous wounds in young and aged individuals.

Epidermal autophagy and beclin 1 regulator 1 and loricrin: a paradigm shift in the prognostication and stratification of the American Joint Committee on Cancer stage I melanomas.

BACKGROUND: The updated American Joint Committee on Cancer (AJCC) staging criteria for melanoma remain unable to identify high-risk stage I tumour subsets. OBJECTIVES: To determine the utility of epidermal autophagy and beclin 1 regulator 1 (AMBRA1)/loricrin (AMLo) expression as a prognostic biomarker for AJCC stage I cutaneous melanoma. METHODS: Peritumoral AMBRA1 expression was evaluated in a retrospective discovery cohort of 76 AJCC stage I melanomas. AMLo expression was correlated with clinical outcomes up to 12 years in two independent powered, retrospective validation and qualification cohorts comprising 379 AJCC stage I melanomas. RESULTS: Decreased AMBRA1 expression in the epidermis overlying primary melanomas in a discovery cohort of 76 AJCC stage I tumours was associated with a 7-year disease-free survival (DFS) rate of 81·5% vs. 100% survival with maintained AMBRA1 (P < 0·081). Following an immunohistochemistry protocol for semi-quantitative analysis of AMLo, analysis was undertaken in validation (n = 218) and qualification cohorts (n = 161) of AJCC stage I melanomas. Combined cohort analysis revealed a DFS rate of 98·3% in the AMLo low-risk group (n = 239) vs. 85·4% in the AMLo high-risk cohort (n = 140; P < 0·001). Subcohort multivariate analysis revealed that an AMLo hazard ratio (HR) of 4·04 [95% confidence interval (CI) 1·69-9·66; P = 0·002] is a stronger predictor of DFS than Breslow depth (HR 2·97, 95% CI 0·93-9·56; P = 0·068) in stage IB patients. CONCLUSIONS: Loss of AMLo expression in the epidermis overlying primary AJCC stage I melanomas identifies high-risk tumour subsets independently of Breslow depth. What's already known about this topic? There is an unmet clinical need for biomarkers of early-stage melanoma. Autophagy and beclin 1 regulator 1 (AMBRA1) is a proautophagy regulatory protein with known roles in cell proliferation and differentiation, and is a known tumour suppressor. Loricrin is a marker of epidermal terminal differentiation. What does this study add? AMBRA1 has a functional role in keratinocyte/epidermal proliferation and differentiation. The combined decrease/loss of peritumoral AMBRA1 and loricrin is associated with a significantly increased risk of metastatic spread in American Joint Committee on Cancer (AJCC) stage I tumours vs. melanomas, in which peritumoral AMBRA1 and loricrin are maintained, independently of Breslow depth. What is the translational message? The integration of peritumoral epidermal AMBRA1/loricrin biomarker expression into melanoma care guidelines will facilitate more accurate, personalized risk stratification for patients with AJCC stage I melanomas, thereby facilitating stratification for appropriate follow-up and informing postdiagnostic investigations, including sentinel lymph node biopsy, ultimately resulting in improved disease outcomes and rationalization of healthcare costs.

Harnessing autophagy to overcome mitogen-activated protein kinase kinase inhibitor-induced resistance in metastatic melanoma.

BACKGROUND: Patients with malignant melanoma often relapse after treatment with BRAF and/or mitogen-activated protein kinase kinase (MEK) inhibitors (MEKi) owing to development of drug resistance. OBJECTIVES: To establish the temporal pattern of CD271 regulation during development of resistance by melanoma to trametinib, and determine the association between development of resistance to trametinib and induction of prosurvival autophagy. METHODS: Immunohistochemistry for CD271 and p62 was performed on human naevi and primary malignant melanoma tumours. Western blotting was used to analyse expression of CD271, p62 and LC3 in melanoma subpopulations. Flow cytometry and immunofluorescence microscopy was used to evaluate trametinib-induced cell death and CD271 expression. MTS viability assays and zebrafish xenografts were used to evaluate the effect of CD271 and autophagy modulation on trametinib-resistant melanoma cell survival and invasion, respectively. RESULTS: CD271 and autophagic signalling are increased in stage III primary melanomas vs. benign naevi. In vitro studies demonstrate MEKi of BRAF-mutant melanoma induced cytotoxic autophagy, followed by the emergence of CD271-expressing subpopulations. Trametinib-induced CD271 reduced autophagic flux, leading to activation of prosurvival autophagy and development of MEKi resistance. Treatment of CD271-expressing melanoma subpopulations with RNA interference and small-molecule inhibitors to CD271 reduced the development of MEKi resistance, while clinically applicable autophagy modulatory agents - including Δ9-tetrahydrocannabinol and Vps34 - reduced survival of MEKi-resistant melanoma cells. Combined MEK/autophagy inhibition also reduced the invasive and metastatic potential of MEKi-resistant cells in an in vivo zebrafish xenograft. CONCLUSIONS: These results highlight a novel mechanism of MEKi-induced drug resistance and suggest that targeting autophagy may be a translatable approach to resensitize drug-resistant melanoma cells to the cytotoxic effects of MEKi.

The prognostic significance and impact of the CXCR4-CXCR7-CXCL12 axis in primary cutaneous melanoma.

BACKGROUND: Expression of the chemokine receptor CXCR4 is known to regulate melanoma metastasis to distant sites with high expression of the CXCL12 ligand. However, the prognostic impact of CXCR4 expression and potential for autocrine-mediated activation of prosurvival mitogen-activated protein kinase signalling remains enigmatic. Furthermore, expression of the decoy receptor CXCR7 within the local cutaneous melanoma microenvironment remains undefined. OBJECTIVES: To define the contribution and prognostic impact of CXCR4-CXCR7-CXCL12 signalling in primary cutaneous melanomas and the immediate tumour microenvironment. METHODS: Immunohistochemical/immunofluorescent expression of CXCR4, CXCR7 or CXC12 was analysed in human metastatic melanoma cell lines, primary cutaneous cell types and a retrospective cohort of primary melanomas/benign naevi. CXCL12 secretion by melanoma/cutaneous cells was evaluated by enzyme-linked immunosorbent assay, and autocrine CXCR4-CXCL12 signalling was investigated by addition of a CXCL12-neutralizing antibody. RESULTS: CXCR4 expression was significantly higher in primary melanomas that subsequently metastasized after 7 years (P = 0·037). Stratification for American Joint Committee on Cancer (AJCC) stage II disease revealed significantly decreased disease-free survival in patients with > 50% CXCR4 expression (P = 0·036), while comparative analysis of CXCL12 expression in the adjacent epidermis of all AJCC stage melanomas revealed increased CXCL12 correlated with prolonged time to metastasis (P = 0·014). CXCR7 was expressed within the primary melanoma microenvironment but was absent on primary tumours. Addition of anti-CXCL12 to BRAF-mutant melanoma cells resulted in downregulation of phospho-CXCR4 and phospho-extracellular signal-related kinase, indicating autocrine CXCR4-CXCL12 signalling. CONCLUSIONS: CXCR4 expression defines a potential prognostic biomarker for AJCC stage II melanoma. Moreover, targeting the CXCR4-CXCR7-CXCL12 axis may represent a novel therapeutic strategy to prevent early melanoma progression.

Inducing apoptosis of cancer cells using small-molecule plant compounds that bind to GRP78.

BACKGROUND: Glucose regulated protein 78 (GRP78) functions as a sensor of endoplasmic reticulum (ER) stress. The aim of this study was to test the hypothesis that molecules that bind to GRP78 induce the unfolded protein response (UPR) and enhance cell death in combination with ER stress inducers. METHODS: Differential scanning calorimetry (DSC), measurement of cell death by flow cytometry and the induction of ER stress markers using western blotting. RESULTS: Epigallocatechin gallate (EGCG), a flavonoid component of Green Tea Camellia sinensis, and honokiol (HNK), a Magnolia grandiflora derivative, bind to unfolded conformations of the GRP78 ATPase domain. Epigallocatechin gallate and HNK induced death in six neuroectodermal tumour cell lines tested. Levels of death to HNK were twice that for EGCG; half-maximal effective doses were similar but EGCG sensitivity varied more widely between cell types. Honokiol induced ER stress and UPR as predicted from its ability to interact with GRP78, but EGCG was less effective. With respect to cell death, HNK had synergistic effects on melanoma and glioblastoma cells with the ER stress inducers fenretinide or bortezomib, but only additive (fenretinide) or inhibitory (bortezomib) effects on neuroblastoma cells. CONCLUSION: Honokiol induces apoptosis due to ER stress from an interaction with GRP78. The data are consistent with DSC results that suggest that HNK binds to GRP78 more effectively than EGCG. Therefore, HNK may warrant development as an antitumour drug.

Inhibition of CXCR4-CXCL12 chemotaxis in melanoma by AMD11070.

BACKGROUND: Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma. The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult. METHODS: AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100. RESULTS: AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100. Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070. CONCLUSION: Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver. Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.

Increasing the therapeutic efficacy of docetaxel for cutaneous squamous cell carcinoma through the combined inhibition of phosphatidylinositol 3-kinase/AKT signalling and autophagy.

Early-stage cutaneous squamous cell carcinoma (cSCC) has a favourable prognosis. Metastatic disease is probably associated with chemoresistance mediated through the activation of pro-survival phosphatidylinositol 3-kinase/AKT signalling. Inhibition of activated AKT partially increases chemosensitivity but induces autophagy, the principal lysosomal mechanism for the bulk degradation and recycling of proteins and damaged organelles. The aim of the current study was to test the hypothesis that combined inhibition of AKT signalling and autophagy by the lysosomal inhibitor chloroquine increases the susceptibility to docetaxel-induced apoptosis of cSCC cells isolated from a lymph-node metastasis. Combined AKT inhibition and chloroquine treatment of MET 4 cSCC cells resulted in significantly enhanced inhibition of cell viability and apoptosis induced by clinically achievable concentrations of docetaxel (P < 0.001). Inhibition of both autophagy and AKT thus represents an effective and viable therapeutic strategy to increase the cytotoxicity of docetaxel for the treatment of advanced cSCC.

NFAT signalling is a novel target of oncogenic BRAF in metastatic melanoma.

BACKGROUND: Metastatic melanoma is the most deadly form of skin cancer and with an overall 5-year survival rate of <11%, there is an acute need for novel therapeutic strategies. Activating mutations in the BRAF oncogene are present in 50-70% of cases and contribute to tumourigenesis, thus, defining downstream targets of oncogenic BRAF may help define novel targets for therapeutic intervention. The Ca(2+)/calcineurin-regulated transcription factor, Nuclear factor of activated T-cells (NFAT), is important in the pathogenesis of several human cancers, target genes of which are also known to contribute to melanoma progression. One such NFAT target gene is COX-2, increased expression of which correlates with poor prognosis; however, upstream regulators of COX-2 in melanoma remain undefined. Therefore, the aim of this study was to evaluate NFAT expression and activity in metastatic melanoma and establish whether or not oncogenic BRAF signalling modulates NFAT activity and determine if NFAT is a key upstream regulator of COX-2 in melanoma. METHODS: Nuclear factor of activated T-cells transcriptional activity and protein expression were determined in three human metastatic melanoma cell lines with differing B-RAF mutational status. NFAT activation by oncogenic BRAF(V600E) was explored by BRAF(V600E) overexpression and application of the specific MEK inhibitor PD98059. Regulation of COX-2 expression by NFAT was investigated using NFAT-targeted siRNA, calcineurin inhibitors cyclosporin A and FK506, in addition to COX-2 luciferase reporter vectors that selectively lacked NFAT binding sites. RESULTS: NFAT transcriptional activity was increased in BRAF-mutated melanoma cells compared with wild-type cells. Furthermore, in wild-type cells, overexpression of BRAF(V600E) increased NFAT activity, which was blocked by the MEK inhibitor PD98059. Using calcineurin inhibitors and siRNA-mediated knockdown of NFAT2 and 4, we show NFAT is required for COX-2 promoter activation and protein induction in metastatic melanoma cells. CONCLUSION: NFAT2 and 4 are expressed in human metastatic melanoma cell lines and are activated by oncogenic BRAF(V600E) via MEK/ERK signalling. NFAT is an important upstream regulator of COX-2 in metastatic melanoma. Furthermore, as the BRAF/MEK/ERK pathway is hyperactive in other malignancies and MEK/ERK are also activated by oncogenic RAS in 30% of all human cancers, the potential to exploit NFAT signalling for therapeutic benefit warrants further investigation.

Apoptosis of N-type neuroblastoma cells after differentiation with 9-cis-retinoic acid and subsequent washout.

BACKGROUND: The overall survival rate for patients with neuroblastoma has improved over the past two decades, but long-term survival for the subgroup of patients with high-risk disease remains low. In recent years, there has been interest in the potential clinical use of drugs able to induce differentiation of neuroblastoma cells. Since 9-cis-retinoic acid induces better and more sustained differentiation of neuroblastoma in vitro than other retinoic acid isomers, this may be a more appropriate retinoid for use in neuroblastoma therapy. PURPOSE: The purpose of this work was to compare the long-term effects of all-trans- and 9-cis-retinoic acid on neuroblastoma differentiation using an N-type (neuroblastic) cell line, SH SY 5Y, as an in vitro model. In addition, we wanted to find out whether 9-cis-retinoic acid would induce programmed cell death (apoptosis) in these N-type neuroblastoma cells and to determine whether the effects of either 9-cis- or all-trans-retinoic acid are dependent on their continued presence in the culture medium. METHODS: SH SY 5Y cells were incubated in either the continued presence of all-trans- or 9-cis-retinoic acid or for 5 days with retinoic acid followed by culture in the absence of retinoid for up to 13 days. Morphologic changes were observed using phase-contrast and scanning electron microscopy. Apoptosis was determined by flow cytometry of propidium iodide-stained cells and by using terminal deoxynucleotidyl transferase to end-label DNA fragments in situ in apoptotic cells. RESULTS: Culture of SH SY 5Y cells with all-trans- or 9-cis retinoic acid for 5 days induced morphologic differentiation and inhibited cell growth. These effects were maintained in the continuous presence of each retinoic acid isomer but were more profound in cells treated with 9-cis-retinoic acid. The differentiation of cells treated with all-trans-retinoic acid was reversible once retinoic acid was removed from the medium. Conversely, apoptosis was induced in cells treated with 9-cis-retinoic acid for 5 days and cultured for 9 days (4 days after washout) but not in cells cultured in the continuous presence of 9-cis-retinoic acid. This effect was specific to 9-cis-retinoic acid. CONCLUSIONS: Previous studies have demonstrated differential responses to all-trans-retinoic acid in N- and S-type (substrate-adherent or Schwann-like) neuroblastoma cells: Apoptosis is induced in S-type cells, whereas differentiation occurs in N-type cells. The present results show that, unlike all-trans-retinoic acid, 9-cis-retinoic acid induces both differentiation and apoptosis in N-type SH SY 5Y neuroblastoma cells. However, apoptosis was dependent on removal of 9-cis-retinoic acid from the culture medium. IMPLICATIONS: Since both differentiation and apoptosis are involved in tumor regression, 9-cis-retinoic acid may be a more appropriate retinoid for clinical trials in neuroblastoma. The dependence of apoptosis on treatment and subsequent removal of 9-cis-retinoic acid implies that drug scheduling may be an important parameter affecting therapeutic efficacy.

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma.

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF(V600E) mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF(V600E) induces a chronic ER stress status directly increasing basal cell autophagy. BRAF(V600E)-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF(V600E)-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF(V600E) melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.

Differential effects of retinoic acid isomers on the expression of nuclear receptor co-regulators in neuroblastoma.

Retinoic acid modulates growth and induces differentiation and apoptosis of neuroblastoma cells in vitro, with the all-trans and 9-cis isomers having different biological properties. Transcriptional activation in response to retinoic acid isomers is mediated by retinoic acid receptors and retinoid X receptors. The differential expression of co-activators and co-repressors which preferentially interact with retinoic acid receptors or retinoid X receptors may be a mechanism leading to different cellular responses to 9-cis and all-trans retinoic acid. To test this hypothesis, we have studied the expression of the nuclear receptor co-regulators TIF1alpha, TIF1beta, SUG1 and SMRT in the N-type and S-type neuroblastoma cell lines SH SY 5Y and SH S EP. Transcripts for all four co-regulators were expressed in these neuroblastoma cells. The expression of TIF1alpha, TIF1beta and SUG1 did not change in response to retinoic acid; however, SMRT was induced in both neuroblastoma cell lines, but particularly by all-trans retinoic acid in SH S EP cells. An additional co-activator, Trip3, was isolated by differential mRNA display and shown to be preferentially induced by 9-cis retinoic acid in SH SY 5Y and SH S EP cells. These data suggest that retinoic acid isomer-specific induction of nuclear receptor co-regulators may determine, in part, the differential biological effects of retinoic acid isomers.

Gene expression and neuroblastoma cell differentiation in response to retinoic acid: differential effects of 9-cis and all-trans retinoic acid.

Retinoic acid has considerable potential for the chemoprevention and chemotherapy of cancer. Neuroblastoma cells differentiate in response to retinoic acid in vitro, an observation that has led to clinical trials using either the 13-cis or all-trans isomers of retinoic acid. We review the effects of retinoic acid on neuroblastoma, and the potential involvement of nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9-cis retinoic acid is a ligand for RXRs, and we review recent data on the differential effects of 9-cis and all-trans retinoic acid on neuroblastoma differentiation and proliferation in vitro, and possible mechanisms of action via hetero- and homodimers of RARs and RXRs. Although there is uncertainty whether or not 9-cis retinoic acid produces its biological effects primarily via RXR homodimers, in vitro data suggest that this isomer of retinoic acid or stable analogues may have considerable potential for the treatment of resistant, disseminated neuroblastoma.

Retinoid-induced differentiation of neuroblastoma: comparison between LG69, an RXR-selective analogue and 9-cis retinoic acid.

The aim of this study was to investigate in vitro the effects of all-trans retinoic acid (RA), 9-cis RA and the RXR-selective analogue, LG69, on the morphological differentiation, proliferation and gene expression of neuroblastoma cells. Three different cell lines were cultured with the retinoid for either 9 continuous days or for 5 days followed by 4 days without the retinoid and morphological differentiation was assessed both qualitatively and quantitatively. SH SY 5Y cell proliferation was examined by measuring cell numbers after exposure to the retinoids and RAR-beta gene expression was examined by Northern blot analysis. Morphological differentiation was more effectively induced by all-trans and 9-cis RA than by LG69. SH SY 5Y cells, when treated with 9-cis RA for only 5 of the 9 days of culture, underwent apoptosis, but this was not seen with 9 days continuous exposure nor with LG69. Inhibition of SH SY 5Y cell proliferation by all-trans or 9-cis RA was dose-dependent, but LG69 had little effect. Conversely, LG69 induced higher expression of RAR-beta than all-trans RA, but less than that produced by 9-cis RA. These data suggest that 9-cis RA as a single agent is the most effective modulator of neuroblastoma behaviour and may be the most appropriate therapeutic agent.

Retinoids in neuroblastoma therapy: distinct biological properties of 9-cis- and all-trans-retinoic acid.

We investigated the potential for 9-cis-retinoic acid in the differentiation therapy of neuroblastoma using an N-type neuroblastoma cell line, SH SY 5Y, as an experimental model. In these cells, 9-cis-retinoic acid is more effective than other isomers at inducing the expression of RAR-beta. An RAR-alpha-specific antagonist inhibited the induction of RAR-beta in response to all-trans-but not to 9-cis-retinoic acid. This indicates that the mechanism of gene induction by 9-cis-retinoic acid differs markedly from all-trans-retinoic acid. 9-cis-retinoic acid is also better than all-trans at producing sustained morphological differentiation and inhibition of proliferation of SH SY 5Y cells. Although N-type neuroblastoma cells are not thought to undergo apoptosis in response to all-trans-retinoic acid, we observed a significant degree of apoptosis in SH SY 5Y cells treated with 9-cis-retinoic acid for 5 days and then cultured in the absence of retinoid, an effect not observed in cells treated with the all-trans isomer. These results suggest that 9-cis- and all-trans-retinoic acid have distinct biological properties and that 9-cis retinoic acid may be clinically effective in neuroblastoma by inducing both differentiation and apoptosis under an appropriate treatment regimen.

Enumeration of lymphocyte subpopulations by immunofluorescent staining of whole blood smears.

A simple and rapid technique for the enumeration of lymphocyte subpopulations by immunofluorescent staining of blood smears is described. The extremely small volumes of blood required make the technique particularly suitable for samples from paediatric patients. Results compare closely with conventional staining of lymphocytes in suspension.

Retinoic acid receptor expression during the in vitro differentiation of human neuroblastoma.

Neuroblastoma cells differentiate in response to retinoic acid and cyclic AMP. We have examined the expression of retinoic acid receptors (RARs) in relation to neuroblastoma differentiation and show that short term exposure of SK N SH, SH SY 5Y AND GI LI N cells to physiological concentrations of retinoic acid results in induction of RAR-beta, particularly the lower transcript. Cyclic AMP has no effect on retinoic acid mediated induction and does not alter RAR expression patterns. These data are discussed in the light of evidence that retinoic acid and cyclic AMP act either on different control pathways or at different points within a common pathway.

Concentration-dependent effects of 9-cis retinoic acid on neuroblastoma differentiation and proliferation in vitro.

The observation that neuroblastoma cells differentiate in response to retinoic acid (RA) in vitro has led to clinical trials using either 13-cis or all-trans RA. Since 9-cis RA may also have important biological functions, we have compared the potential of RA isomers to induce differentiation and inhibit cell proliferation of SH SY 5Y neuroblastoma cells. 9-cis RA at high concentrations is better at inducing morphological differentiation than either all-trans or 13-cis RA and as effective at inhibiting proliferation. Hence, 9-cis RA or stable analogues may have important therapeutic potential in the treatment of neuroblastoma.

The effects of anesthesia and surgery on lymphocyte populations and function in infants and children.

A study was designed to test the hypothesis that the lymphopenia caused by surgical stress in children may arise through selective depletion of one or more lymphocyte subsets. Blood samples from 22 children were taken pre- and postoperatively and 6, 12, 24, and 48 hours after surgery. Lymphocyte subsets were identified and counted using monoclonal antibodies and indirect immunofluorescence. By six hours postoperatively, the mean total lymphocyte count had fallen by 1.87 x 10(9)/L (P less than .01); this was largely due to the fall in helper T cells (1.53 x 10(9)/L, P less than .01) and both counts remained depressed for at least 48 hours. The helper:suppressor ratio also fell, from 3.42 to 1.92 (P less than .01), but had recovered by 48 hours. Lymphocyte function as measured by the response to pokeweed mitogen and concanavalin A was also reduced six hours postoperatively. These changes were independent of age. Major surgery in infants and children causes a selective reduction in helper T lymphocyte numbers, helper:suppressor ratio, and lymphocyte function. This suggests that immune competence in the immediate postoperative period in children is reduced, as it is in adults. The duration of this and its relationship to infection are not yet known.