SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma.

BACKGROUND: We previously demonstrated an inverse correlation between tyrosinase-related protein 1 (TYRP1) mRNA expression in melanoma metastases and patient survival. However, TYRP1 protein was not detected in half of tissues expressing mRNA and did not correlate with survival. Based on a study reporting that 3' untranslated region (UTR) of TYRP1 mRNA contains two miR-155-5p (named miR-155) binding sites exhibiting single-nucleotide polymorphisms (SNPs) that promote (matched miRNA-mRNA interaction) mRNA decay or not (mismatched), we aimed to investigate the role of miR-155 in the regulation of TYRP1 mRNA expression and protein translation accounting for these SNPs. METHODS: The effect of miR-155 on TYRP1 mRNA/protein expression was evaluated in two melanoma cell lines harbouring matched or mismatched miR-155-TYRP1 mRNA interaction after transfection with pre-miR-155. In parallel, 192 skin and lymph node melanoma metastases were examined for TYRP1 mRNA/protein, miR-155 and SNPs and correlated with patient survival. TYRP1 mRNA, SNPs at its 3'UTR and miR-155 were analysed by RT-qPCR, whereas TYRP1 protein was evaluated by western blot in cell lines and by immunohistochemistry in metastatic tissues. RESULTS: The miR-155 induced a dose-dependent TYRP1 mRNA decay and hampered its translation into protein in the line with the 'match' genotype. In melanoma metastases, TYRP1 mRNA inversely correlated with miR-155 expression but not with TYRP1 protein in the 'match' group, whereas it positively correlated with protein but not with miR-155 in the 'mismatch' group. Consequently, in the latter group, TYRP1 protein inversely correlated with survival. CONCLUSION: Polymorphisms in 3'UTR of TYRP1 mRNA can affect TYRP1 mRNA regulation by miR-155 and its subsequent translation into protein. These SNPs can render TYRP1 mRNA and protein expression nonsusceptible to miR-155 activity and disclose a prognostic value for TYRP1 protein in a subgroup of melanoma patients. These data support the interest in the prognostic value of melanogenic markers and propose TYRP1 to refine prognosis in patients with advanced disease.

Tyrosinase-related protein 1 mRNA expression in lymph node metastases predicts overall survival in high-risk melanoma patients.

BACKGROUND: Clinical outcome of high-risk melanoma patients is not reliably predicted from histopathological analyses of primary tumours and is often adjusted during disease progression. Our study aimed at extending our previous findings in skin metastases to evaluate the prognostic value of tyrosinase-related protein 1 (TYRP1) in lymph node metastases of stages III and IV melanoma patients. METHODS: TYRP1 mRNA expression in 104 lymph node metastases was quantified by real-time PCR and normalised to S100 calcium-binding protein B (S100B) mRNA expression to correct for tumour load. TYRP1/S100B ratios were calculated and median was used as cutoff value. TYRP1/S100B mRNA values were correlated to clinical follow-up and histopathological characteristics of the primary lesion. RESULTS: A high TYRP1/S100B mRNA ratio significantly correlated with reduced disease-free (DFS) and overall survival (OS; Cox regression analysis, P=0.005 and 0.01, respectively), increased Breslow thickness (Spearman's rho test, P<0.001) and the presence of ulceration (Mann-Whitney test, P=0.02) of the primaries. Moreover, high TYRP1/S100B was of better prognostic value (lower P-value) for OS than Breslow thickness and ulceration. Finally, it was well conserved during disease progression with respect to high/low TYRP1 groups. CONCLUSION: High TYRP1/S100B mRNA expression in lymph node metastases from melanoma patients is associated with unfavourable clinical outcome. Its evaluation in lymph node metastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lesions and may allow different management of the two groups of patients.

[Survey concerning exposure of staff to UV radiation at four Brussels hospitals]. / Enquête sur l'exposition aux rayons ultraviolets parmi le personnel de quatre hôpitaux bruxellois.

BACKGROUND: The incidence of melanoma has been increasing for 50 years. Exposure to ultraviolet (UV) radiation constitutes the main risk factor. The aim of this study was to analyze the impact on hospital staff behaviour with regard to UV of screening campaigns initiated in Belgium 11 years ago. PATIENTS AND METHODS: We performed a multicentre, before-after study by sending an anonymous survey to the staff of four hospital in Brussels, from March 2010 to April 2010 (group 2: n=895). Demographic, clinical and behavioural data were collected and compared to those collected 23 years ago in the same hospitals (group 1: n=2410). RESULTS: Phototypes in both groups were similar. In group 2, the distribution of naevi tended to be spread over the whole body and the severity of sunburn had decreased. Group 2 participants reported a reduction in active sun exposure, especially in the past 10 years, with less leisure-time tanning. There was a significant increase in holidays in sunny locations, although vacation time was shorter, with prolonged daily and annual exposure. Sunscreens were more frequently used and there was an increase in sun-bed use, especially in beauty parlours. CONCLUSION: Our study comprises a double snapshot of a population of hospital workers at an interval of 23 years. The information and screening campaigns do not seem to have had the desired effect on the hospital staff surveyed. Sunscreen use has in fact resulted in extended UV exposure and the observed exposure pattern is that most frequently involved in melanoma development.

TYRP1 mRNA expression in melanoma metastases correlates with clinical outcome.

BACKGROUND: Clinical outcome of patients with high-risk melanoma cannot be reliably predicted on the basis of classical histopathological examination. Our study aimed to determine in melanoma metastases a gene expression profile associated with patient survival, and to identify and validate marker(s) of poor clinical outcome. METHODS: Skin and lymph node metastases from melanoma patients (training population) were used to identify candidate prognostic marker(s) based on DNA microarray analysis. Additional skin metastases (validation population) were used to assess the prognostic value of the first ranked gene by real-time PCR. RESULTS: We performed microarray analysis in the training population and generated a list of 278 probe sets associated with a shorter survival. We used the first ranked gene, tyrosinase-related protein 1 (TYRP1), further measured its expression in the validation population by real-time PCR and found it to be significantly correlated with distant metastasis-free survival (DMFS), overall survival (OS) and Breslow thickness. We also found that it was fairly well conserved in the course of the disease regardless of the delay to metastasis occurrence. Finally, although Tyrp1 protein (immunohistochemistry (IHC)) was only detected in about half of the samples, we showed that its expression also correlated with Breslow thickness. CONCLUSION: Our data indicate that TYRP1 mRNA expression level, at least in skin metastases, is a prognostic marker for melanoma, and is particularly useful when prognostic pathology parameters at the primary lesion are lacking. Its conserved expression further supports its use as a target for therapy.

Outcomes for and risk factors associated with vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Enterococcus faecium bacteremia in cancer patients.

OBJECTIVE: Vancomycin-resistant enterococci (VRE) are a major cause of nosocomial infection. We sought to compare vancomycin-resistant (VR) Enterococcus faecalis bacteremia and VR Enterococcus faecium bacteremia in cancer patients with respect to risk factors, clinical presentation, microbiological characteristics, antimicrobial therapy, and outcomes. METHODS: We identified 210 cancer patients with VRE bacteremia who had been treated between January 1996 and December 2004; 16 of these 210 had VR E. faecalis bacteremia and were matched with 32 patients with VR E. faecium bacteremia and 32 control patients. A retrospective review of medical records was conducted. RESULTS: Logistic regression analysis showed that, compared with VR E. faecalis bacteremia, VR E. faecium bacteremia was associated with a worse clinical response to therapy (odds ratio [OR], 0.3 [95% confidence interval (CI), 0.07-0.98]; P=.046) and a higher overall mortality rate (OR, 8.3 [95% CI, 1.9-35.3]; P=.004), but the VRE-related mortality rate did not show a statistically significant difference (OR, 6.8 [95% CI, 0.7-61.8]; P=.09). Compared with control patients, patients with VR E. faecalis bacteremia were more likely to have received an aminoglycoside in the 30 days before the onset of bacteremia (OR, 5.8 [95% CI, 1.2-27.6]; P=.03), whereas patients with VR E. faecium bacteremia were more likely to have received a carbapenem in the 30 days before the onset of bacteremia (OR, 11.7 [95% CI, 3.6-38.6]; P<.001). In a multivariate model that compared patients with VR E. faecium bacteremia and control patients, predictors of mortality included acute renal failure on presentation (OR, 15.1 [95% CI, 2.3-99.2]; P=.004) and VR E. faecium bacteremia (OR, 11 [95% CI, 2.7-45.1]; P<.001). No difference in outcomes was found between patients with VR E. faecalis bacteremia and control patients. CONCLUSIONS: VR E. faecium bacteremia in cancer patients was associated with a poorer outcome than was VR E. faecalis bacteremia. Recent receipt of carbapenem therapy was an independent risk factor for VR E. faecium bacteremia, and recent receipt of aminoglycoside therapy was independent risk factor for E. faecalis bacteremia.

Bronchopulmonary dysplasia and pulmonary hypertension: a meta-analysis.

OBJECTIVE: Pulmonary hypertension (PH) is a complication of bronchopulmonary dysplasia (BPD) but the true impact of PH in patients with BPD remains unclear. We sought to systematically review and meta-analyze incidence of PH in BPD and compare clinical outcomes of BPD patients with PH to those without PH in preterm infants. STUDY DESIGN: Medline, Embase, PsychINFO and CINAHL were searched from January 2000 through December 2015. Cohort, case-control and randomized studies were included. Case-reports, case-series and letters to editors and studies with high risk of bias were excluded. Study design, inclusion/exclusion criteria, diagnostic criteria for BPD and PH and outcomes were extracted independently by two co-authors. RESULTS: The pooled incidence of PH in patients with BPD (any severity) was 17% (95% confidence interval (CI) 12 to 21; 7 studies) and 24% (95% CI 17 to 30; 9 studies) in moderate-severe BPD. Patients with BPD have higher unadjusted odds of developing PH compared to those without BPD (odds ratio (OR) 3.00; 95% CI 1.18 to 7.66; 4 studies). Patients with BPD and PH were at higher odds of mortality (OR 5.29; 95% CI 2.07 to 13.56; 3 studies) compared with BPD without PH, but there was no significant difference in duration of initial hospitalization, duration of supplemental oxygen requirement or need for home oxygen. No studies included in this review reported on long-term pulmonary or neurodevelopmental outcomes. CONCLUSIONS: PH occurs in one out of 4 to 5 preterm neonates with BPD. Patients with BPD and PH may have higher odds of mortality; however, there is urgent need for high quality studies that control for confounders and provide data on long-term outcomes.

A simple and accurate new method for cytostatics dosimetry in isolation perfusion of the limbs based on exchangeable blood volume determination.

Current methods for cytostatic dosimetry in isolation perfusion of the limbs are based on either limb tissue volume (LTV) or body weight. None of them take into account the actual blood volume intra- and extracorporal, including even the blood leakage if any, in which the pharmacokinetics take place. The present study describes a method which allows the assessment of the actual exchangeable blood volume. The latter is calculated by a formula based on three hematocrit measurements. Thirty-one cases entered the study. Exchangeable limb blood volume representing the limb vascular bed was found to average 340 +/- 148 (SD) ml for upper limb perfusion and 768 +/- 279 and 621 +/- 454 ml for iliac and femoropopliteal perfusion, respectively. There was a good correlation between exchangeable limb blood volume and limb tissue volume (LTV, r = 0.7), a poor one with body weight (r = 0.3), and no correlation at all with body surface. Melphalan dosage was calculated per ml of blood and applied at 20 to 40 micrograms/ml. Comparison between calculated dose and concentration measured by high performance liquid chromatography showed a high correlation (r = 0.963). Since there was a correlation between exchangeable limb blood volume and LTV, it was possible to derive a conversion for melphalan dosage where 13 mg/liter corresponds to 20 micrograms/ml in upper limb perfusion and 10 mg/liter corresponds to 40 micrograms/ml in lower limb perfusion. Comparison between calculated melphalan dosage based on our method and the LTV method showed a large dispersion of values in the latter (12 to 18% coefficient of variation) while the dispersion given by the body weight-based method increased 2-fold (16 to 31% coefficient of variation). It is concluded that the present dosimetry method is the most suitable up to the present for accurate prediction of cytostatic concentration in isolation perfusion.

Mucolytic Agents Can Enhance HER2 Receptor Accessibility for [(89)Zr]Trastuzumab, Improving HER2 Imaging in a Mucin-Overexpressing Breast Cancer Xenograft Mouse Model.

PURPOSE: Binding of trastuzumab to HER2 receptors can be impaired by steric hindrance caused by mucin MUC4. As mucolytic drugs can breakdown disulfide bonds of mucoproteins, we checked if this approach could positively affect zirconium-89-labeled trastuzumab ([(89)Zr]T) binding/uptake. PROCEDURES: The effect of N-acetylcysteine (NAC) and MUC4 knockdown/stimulation on [(89)Zr]T binding/uptake were evaluated in MCF7(HER2-), BT474 and SKBr3(HER2+/MUC4-), and JIMT1(HER2+/MUC4+) cell lines. The results were then validated in SKBR3 and JIMT1 tumor-bearing nude mice with a microPET-CT and ex vivo analysis. RESULTS: Significant increases in [(89)Zr]T binding/uptake were observed in JIMT1 cells following MUC4 knockdown (62.4 ± 6.5%) and exposure to NAC (62.8 ± 19.4%). Compared to controls, mice treated with NAC showed a significant increase in [(89)Zr]T uptake in MUC4 tumors on microPET-CT (SUVmean (18.3 ± 4.7%), SUVmax (41.7 ± 8.4%)) and individual organ counting (37.3 ± 18.3%). In contrast, no significant differences were observed in SKBr3. CONCLUSION: NAC can enhance [(89)Zr]T accumulation and improve the HER2 imaging of MUC4-overexpressing tumors. The potential positive impact on trastuzumab-based treatment deserves further investigation.

Transcriptional repression of the microphthalmia gene in melanoma cells correlates with the unresponsiveness of target genes to ectopic microphthalmia-associated transcription factor.

In the melanocyte, expression of genes required for pigment formation is mediated by the microphthalmia transcription factor, which is also critical for the development and survival of normal melanocytes during embryogenesis. Here we show that the expression of the melanocyte-specific isoform of microphthalmia transcription factor is lost in a subset of human melanoma cell lines, accompanied by the repression of tyrosinase and tyrosinase-related proteins 1 and 2, the three transcriptional target genes for microphthalmia. After the forced expression of microphthalmia transcription factor in melanoma cells where the expression of endogenous microphthalmia gene was found to be extinguished, no restoration of the melanogenic phenotype occurred and the transcription of the three microphthalmia transcription factor target genes remained silent. The transcription activation domain of microphthalmia transcription factor, tested as a GAL-MITF fusion protein, remained fully functional in these cells, however, and ectopic microphthalmia transcription factor localized normally to the nucleus and bound to the tyrosinase initiator E-box in gel retardation assays. Thus, the block of differentiation in microphthalmia-transcription-factor-negative melanomas extended the transcriptional repression of the microphthalmia transcription factor gene alone, and endogenous promoters in these melanoma cells became no longer responsive to microphthalmia transcription factor when this was substituted exogenously. The data presented suggest that a specific nuclear context is required for the transcriptional activation of the melanocyte markers by the microphthalmia transcription factor in malignant melanocytes and this specificity is lost concomitantly with the transcriptional repression of microphthalmia transcription factor.

Comparison of HPLC and stereologic image analysis for the quantitation of eu- and pheomelanins in nevus cells and stimulated melanoma cells.

The aim of the study was to compare two methods of quantitating eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy, and stereology. In a previous study, we showed good correlations between both methods for total melanin but not for eumelanins or pheomelanins. We describe here the same comparison in more pigmented cells (nevus cells and stimulated HBL melanoma cells). Transmission electron microscopy micrographs were image analyzed to generate several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanin content, and the number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results showed a good correlation between both methods for total melanin, eumelanins, and pheomelanins with an r equal to 0.99, 0.91, and 0.93, respectively, when all the points were used in the linear regression analyses. In the melanoma cell group (HBL cells cultured in media of different compositions), the chemical and morphometric estimations were not parallel in the case of eumelanins and pheomelanins. In addition, the stereologic and high performance liquid chromatography pheomelanins to eumelanins ratios were still not correlated. These results demonstrate the relevancy of the stereologic method, but the low level of melanization, the possible lack of specificity of melanogenesis in melanoma cells, and a problem of sensitivity of the stereologic method in this context seem to be obstacles in obtaining better results. The utilization of normal human melanocytes could give some answers to our hypotheses.

Glutathione depletion increases tyrosinase activity in human melanoma cells.

The aim of the present work was to estimate the effect of intracellular glutathione depletion on melanogenesis in human melanoma cells. We determined tyrosine hydroxylation activity, the rate-limiting step of the pathway, and 14C-melanin formation, an assay reflecting the global eumelanogenic pathway. Intracellular glutathione was depleted by treatment with buthionine-S-sulfoximine, a well-known inhibitor of gamma-glutamylcysteine synthetase. The intracellular depletion of glutathione was substantial after 20 h of incubation with 50 microM buthionine-S-sulfoximine, although a significant effect could be observed after 6 h. Tyrosine hydroxylase activity increased in parallel with glutathione depletion, to reach 160% with respect to the control values during 24 h of buthionine-S-sulfoximine treatment. We have found the response to buthionine-S-sulfoximine to be dose dependent and the two different human cell lines HBL and LND1 to have similar, if not identical, responses. 14C-melanin formation assay revealed even greater activation, up to 400% of the control values. This indicates that glutathione depletion may have two distinct effects: first, a direct one on tyrosinase activity and, second, an effect on the promotion of eumelanogenesis. The stimulation of tyrosine hydroxylase can be explained by a possible inactivation of the enzyme by endogenous thiol compounds rather than by a direct effect of buthionine-S-sulfoximine itself on tyrosinase. The data suggest that thiol compounds may play a role for stimulation of melanogenesis by ultraviolet radiation.

Cysteine deprivation promotes eumelanogenesis in human melanoma cells.

Melanocytic cells can produce two types of pigment, pheomelanin or eumelanin. We used two types of human melanoma cell lines to explore the regulation of pigmentation by biochemical and enzymatic studies. These two cell lines were previously designated as either pheomelanotic or of mixed type when cultured in a medium rich in cysteine. We analyzed the effects of L-cysteine depletion on melanin synthesis and the involvement of the tyrosinase-related proteins in the production of both eumelanin and pheomelanin. Cultures were exposed to L-cysteine concentrations ranging from 206 to 2.06 microM, and the following parameters were measured: tyrosine hydroxylase activity, intracellular L-cysteine and glutathione concentrations, eumelanin and pheomelanin formation, and tyrosinase-related protein-1 and -2 mRNA levels. Extracellular L-cysteine depletion significantly increased tyrosine hydroxylase activity and promoted both eumelanogenesis and visible pigmentation in both lines. In contrast, pheomelanogenesis was increased only in the pheomelanotic cell line. Whereas eumelanogenesis was apparent upon L-cysteine depletion, tyrosinase-related protein-1 expression was not induced in the pheomelanotic cells, and tyrosinase-related protein-2 expression remained unchanged. Thus, tyrosinase-related protein-1 mRNA expression seems to be concomitant with eumelanogenesis when the L-cysteine concentration is high, but does not appear essential for eumelanogenesis at low L-cysteine concentrations. The mechanisms governing pheomelanin to eumelanin balance are dependent on L-cysteine, glutathione, and tyrosinase-related protein-1 expression, but none of these factors alone appears to be dominant in directing the synthesis of a particular type of melanin.

Alpha-melanocyte-stimulating hormone inhibits NF-kappaB activation in human melanocytes and melanoma cells.

Alpha-melanocyte-stimulating hormone is produced by several different cell types including neural cells, endothelial cells, monocytes, and keratinocytes. A biologic role in melanocyte pigmentation is widely recognized, but more recent studies describe a part in modulating inflammatory and immune responses. The aim of the this study was to investigate the mechanism by which alpha-melanocyte-stimulating hormone antagonizes proinflammatory cytokine action. We report that alpha-melanocyte-stimulating hormone (10-9 M) was effective in opposing a tumor necrosis factor-alpha stimulated increase in NF-kappaB DNA binding activity in: (i) normal ocular melanocytes; (ii) cells cultured from ocular melanoma tumors; and (iii) two cutaneous melanoma cell lines. NF-kappaB is activated by many inflammatory mediators and controls transcription of genes required for immune and inflammatory responses. The transcription factor complex was positively identified as the p50/p65 heterodimer, recognized to have transcriptional activating potential. Maximum reduction of NF-kappaB DNA binding activity with alpha-melanocyte-stimulating hormone was detected 2 h after cellular stimulation and varied from between 53% and 18% depending on cell type. Whereas the acute inhibitory effects could be mimicked by elevating cyclic adenosine monophosphate, alpha-melanocyte-stimulating hormone was not found to have any effect on the relative level of IkappaBalpha protein expression over 24 h. These data show that alpha-melanocyte-stimulating hormone has a pronounced effect on NF-kappaB activity in melanocytes and melanoma cells, identifying a specific dimeric complex, and suggest this to be a key pathway by which immunomodulation/anti-inflammation may operate. The results may also be considered in the broader context of general inflammatory pathologies concerning cells which express alpha-melanocyte-stimulating hormone receptors and utilize the NF-kappaB signaling pathway.

TRP-1 expression correlates with eumelanogenesis in human pigment cells in culture.

We have investigated the relationship in human cultured normal and malignant melanocytes between the accumulation of mRNAs encoding tyrosinase and tyrosinase-related protein-1 (TRP-1), the activity of tyrosinase and the presence of melanin. Tyrosinase mRNA correlates with tyrosinase activity and with the presence of pheomelanin, eumelanin or both melanin types. In contrast TRP-1 mRNA is only detectable in cells containing eumelanin, which suggests a role for TRP-1 in the eumelanin synthesis pathway.

alpha-Melanotropin immunoreactivity in human melanoma exudate is related to necrosis.

We have previously reported high immunoreactive alpha-MSH (IR-alpha-MSH) concentrations in melanoma patients' plasma, as well as significant amounts in melanoma metastases and cells grown in culture. Necrosis within the melanoma tumour leads to a massive proteolysis of intracellular proteins and release of cell content: this might significantly contribute to the elevated IR-alpha-MSH plasma levels measured in melanoma patients. To test this hypothesis, we studied the necrosis-related release of MSH from human melanoma cells, using a specific radioimmunoassay. The studies of fine-needle biopsies indicated that most of the human melanoma tumour exudates tested contained very high MSH concentrations (> 500 pg/ml; 14/15), while plasma levels were generally normal (< or = 25 pg/ml; 10/15). The level in an exudate from a non-melanoma tumour type was < 40 pg/ml. In vitro studies showed that release of the IR-alpha-MSH was time- and temperature-dependent, and related to cell death.

Comparison and evaluation of different methods for alpha-MSH labelling.

We have studied the behaviour of 125I-labelled alpha-MSH under different experimental conditions. Until now, the chloramine T method had been used by most investigators with variable results. We have tested three other labelling techniques based on 125I mild oxidation: (1) an enzymatic method with lactoperoxidase, (2) a sparingly soluble chloramine method (T.D.G.U.) and (3) modified chloramine T procedure, 'the iodine volatilization method'. Labelled hormone obtained after each kind of iodination was assayed for immunoreactivity. In addition, time course degradation was measured by classical RIA incubation procedures. Charcoal-dextran was used to separate bound and free antigen. We have found chloramine T-iodinated alpha-MSH to be significantly more damaged than preparations obtained by other methods and to be less stable when stored at -18 degrees C. No differences were found between the differently labelled 125I-labelled alpha-MSH fresh preparations in binding to surface receptors of human melanoma cell lines in culture.

Tumor type and vascularity: important variables in infusional brachytherapy with colloidal 32P.

PURPOSE: This study investigated the role of histologic tumor characteristics, in comparison with a normal tissue, and of tumor vascularization on the uptake and retention of colloidal 32P used in infusional brachytherapy of solid cancers. The cytotoxicity of colloidal 32P was also evaluated for two tumors of different radiosensitivity, a melanoma, and a squamous cell carcinoma. METHODS AND MATERIALS: An in vitro analysis of colloidal 32P uptake was carried out on a human melanoma cell line, HBL, a human squamous cell carcinoma, SCC1, and normal fibroblasts, F-NBB. Tumor retention of colloidal 32P was studied in vivo for the HBL and the SCC1 tumors implanted subcutaneously in nude mice. Tumor vascular density was determined by microscopic study of Masson's trichrome slides of HBL and SCC1 tumors of about 1 cm diameter. RESULTS: In vitro studies showed that the time required for maximal cell uptake of colloidal 32P was only 10-20 min for the SCC1 and HBL tumors, while it took at least 60 min for the fibroblasts. After intratumoral injection of macroaggregated albumin (MAA), followed by 50 microCi of colloidal 32P, Bremsstrahlung imaging performed at 6 and 24 h showed that the activity remained in the HBL tumor while some of the radiocolloids leaked from the SCC1 tumor and was trapped in the reticuloendothelial system of the liver. Organ activity counting confirmed this finding: 32P activity was three to four times higher in the HBL than in the SCC1 tumor, whereas the activity in the liver, insignificant in the HBL mice (less than 0.1 microCi/g), was as high as 24 microCi/g in the SCC1 mice. This phenomenon may be explained by the difference in tumor vascular density, estimated for the HBL to be about four times less than that of the SCC1 tumor (5.7 vs. 21.4 blood vessels per mm2 for the HBL and the SCC1 tumors, respectively). CONCLUSION: Intratumoral infusion of colloidal 32P may be a useful complement of radiation therapy in the treatment of nonresectable but accessible solid tumors. Tumor vascularization must be taken into account for a successful vascular blockade by MAA prior to the infusion of colloidal 32P.

Dynamic 131I-labelled insulin distribution in rabbits as seen by in-vivo scintigraphic studies.

The kinetics of 131I-labelled insulin distribution in heart, liver, kidneys and urinary bladder of rabbits were studied in vivo by gamma-camera techniques combined with plasma measurements (glucose concentrations and chromatographic separation of insulin and its degradation products). The distribution space of radioiodinated insulin differed from the vascular bed delineated by radioiodinated serum albumin. During a 20-min gamma-camera recording, radioactive degradation products only appeared in the plasma after 10-12 min. Previous administration of a 10 000-fold excess of unlabelled insulin and 5 ml glucose (20%) did not modify the evolution of 131I-labelled insulin cardiac invasion and the subsequent linear decrease of radioactivity. Conversely, wash-out of radioactivity from the liver and kidney was accelerated after preadministration of this excess of unlabelled hormone, binding in these organs accounting for this acceleration. Urinary bladder filling was imaged later than cardiac, hepatic or renal labelling and was only accelerated by polyuria induced by glucose injection, independent of preadministration of unlabelled hormone.

The degree of pigmentation modulates the radiosensitivity of human melanoma cells.

The relationship between cell pigmentation and radiosensitivity was investigated in two selected human melanoma cell lines with different melanin content (mixed type: eumelanin and pheomelanin, and pheomelanotic phenotypes). The same study was also done after stimulation of melanogenesis (1) by addition of the melanin precursor l-tyrosine to each of the cell lines separately and (2) by irradiation alone with doses ranging from 0 to 10 Gy. We found that a decrease in cell radiosensitivity was correlated with the type of melanin, with a clear involvement of eumelanin rather than pheomelanin. Increasing the intracellular content of both melanins promoted the growth of irradiated cells. Moreover, at a dose of 10 Gy, both tyrosinase activity and melanin cell content were significantly increased in the absence of any other melanogenesis promoter. Our data suggest that the amount of intracellular melanin is inversely related to the radiosensitivity of melanoma cells and may explain at least in part the controversial responses to ionizing radiations reported for melanoma.

Partial characterization of IR-alpha-MSH peptides found in melanoma tumors.

Our previous work indicated that IR-alpha-MSH (immunoreactive alpha-melanocyte-stimulating hormone) plasma levels are three times as high in melanoma patients with progressing disease than in disease-free patients, and that the melanoma tumor itself may be the source of IR-alpha-MSH. Further identification of the material in tumor extracts has been carried out in this study, and the results presented here show that the immunoreactivity is associated with a major fraction of about 16 kDa and another of 5-9 kDa. Significant amounts of the immunoreactive material were also found in human melanoma cells but not in culture supernatants. The presence of this material may be related to the melanogenic status of the tumor cells. We have estimated the intracellular IR-alpha-MSH to be within a 0.4 to 2.3 nM range in melanoma tumor cells. We have investigated the melanogenic effect of the IR-alpha-MSH material and its relationship to alpha-MSH. Purified extracts both from metastases and cultured cells were found to promote frog skin darkening as well as tyrosinase activity in Cloudman S91 melanoma cells. The IR material could also displace labeled alpha-MSH from its binding sites in human melanoma cells. Our data clearly indicate that melanoma cells engage in an autocrine production of alpha-MSH-like bioactive peptides by melanoma cells, of larger mol.wt., which are able to bind to MSH receptors. These peptides may be involved in the regulation of melanogenesis and possibly in the growth and proliferation of melanoma cells by an autocrine/paracrine mechanism.