Genetic landscape of indolent and aggressive Kaposi sarcomas.

BACKGROUND: Kaposi sarcoma (KS) is a rare skin tumour caused by herpesvirus 8 infection and characterized by either indolence or an aggressive course necessitating systemic therapies. The genetic basis of this difference remains unknown. OBJECTIVES: To explore the tumour mutational burden in indolent and aggressive KS. METHODS: We performed whole-exome sequencing on a cohort of 21 KS patients. We compared genetic landscape including tumor mutational burden between the two forms of indolent and agressive KS. RESULTS: Aggressive KS tumours had a significantly higher TMB and a larger cumulative number of deleterious mutations than indolent KS tumours. In addition, all aggressive tumours had at least three deleterious mutations, whereas most indolent tumours harboured only one or no predicted deleterious mutations. Deleterious mutations listed in the Cancer Gene Census were detected exclusively in patients with aggressive disease. An analysis of somatic copy-number alterations (SCNA) revealed a tendency towards higher number of alterations in aggressive KS. CONCLUSIONS: These data suggest that SCNA alterations and an increase in mutational burden promote aggressive KS and that it might be more appropriate to consider indolent KS as an opportunistic skin disease rather than a cancer.

Population biology of the little gulper shark Centrophorus uyato in Lebanese waters.

A total of 38 individuals of the heavily exploited little gulper shark Centrophorus uyato were collected from Lebanese coastal waters using bottom longlines and trammel nets of different meshes at depths ranging from 115 to 600 m between May 2013 and February 2014. Their total lengths were between 45 and 94 cm and their total mass was from 870 to 6700 g. The sex ratio was not significantly different from 1:1, with 20 males and 18 females, but bathymetric sexual segregation occurred. Catch per net setting (CNS) was used as a relative abundance index to detect spatial distribution; trammel nets showed largest CNS ranging from 4·9 to 5·45 kg per unit effort in the north and south, at depths from 120 to 200 m, during spring and summer. The mass-length relationships demonstrated negative allometric growth (b < 3) (males: MT = 0.3585LT2·071 , r2 = 0·94; females: MT = 0.0239LT2·735 , r2 = 0·64). The condition factor as well as the gonado-somatic and hepato-somatic indices of C. uyato in the study area were also calculated. The results are discussed in relation to the distribution, growth and reproduction as well as the management of C. uyato.

Population biology of an endangered species: the common guitarfish Rhinobatos rhinobatos in Lebanese marine waters of the eastern Mediterranean Sea.

This study focuses on the population biology of the common guitarfish Rhinobatos rhinobatos, a cartilaginous fish listed as Endangered in the International Union for the Conservation of Nature (IUCN) Red List. Between December 2012 and January 2014, 67 individuals were collected by bottom longlining in coastal Lebanese marine waters at different ports at depths ranging from 10 to 110 m. The total length (L(T)) of the specimens ranged from 50 to 143 cm, and the mean ± s.d. was 76.2 ± 19.7 cm. The most common L(T) classes were between 60 and 70 cm. The total mass of the specimens ranged from 410 to 10,000 g, and the mean ± s.d. was 1841 ± 1987 g. A total of 34 males and 33 females were collected, and the sex ratio was not significantly different from 1:1. The mass and L(T) relationship showed positive allometric growth (b = 3.096 and r(2) = 0.99), and the mean ± s.d. L(T) at which 50% of the individuals were sexually mature was 84.73 ± 5.81 cm for females and 78.57 ± 4.88 cm for males. The gonado-somatic and hepato-somatic indices were determined along with a condition factor, and parturition appeared to occur in winter. The primary prey items found in the fish stomachs during the autumn and winter seasons were Penaeidae. The results of this study will help to parameterize models of the population dynamics for this exploited fish stock to ensure the long-term sustainability of its fishery.

GEMOX regimen in the treatment of metastatic differentiated refractory thyroid carcinoma.

Treatment options for radioiodine resistant metastatic thyroid cancer patients are limited, and chemotherapy is considered an outdated therapeutic method for differentiated thyroid carcinoma. In this study, we evaluated the activity and safety of gemcitabine and oxaliplatin combination which is considered an out of label therapeutic method in patients with differentiated metastatic thyroid cancer refractory to 131-I treatment. Fourteen refractory patients (8 papillary, 6 follicular), six men/eight women with median age of 63 years and performance status (0-3) were included. Patients received gemcitabine (1,000 mg/m(2)) plus oxaliplatin (100 mg/m(2)) every 2 weeks until 12-cycles and each cycle correspond to 2 weeks treatment. This protocol was approved by the local Institutional Review Boards. Response rate was assessed every four cycles. Progression-free and overall survivals were calculated. Median treatment was 9.5 cycles (range 2-17) with 22 weeks duration. Overall response rate was 57%, with 7% achieving a complete response (1/14), 50% a partial response (7/14), and 28% with a stable disease. All patients with follicular subtype showed objective responses. Eleven patients progressed at a median time of 10.1 months; 10 of 14 patients still alive and the median survival was not reached (median follow-up of 19.8 months). The combination was generally well tolerated. No deaths occurred due to therapy and no grade IV toxicity was recorded. The most common treatment-related adverse events grade 1/3 includes asthenia, peripheral neuropathy, diarrhea, anemia, thrombocytopenia, and neutropenia. In conclusion, the GEMOX regimen is well tolerated and effective in advanced differentiated thyroid cancer. However, this retrospective data on a small sample size are considered preliminary and needs to be evaluated prospectively in a higher number of patients in a clinical trial.

Serum caspase-1 levels in metastatic melanoma patients: relationship with tumour burden and non-response to biochemotherapy.

Interleukin-1beta converting enzymes (ICEs/caspases) are involved in programmed cell death (apoptosis). This study sought to quantify the caspase-1 level in metastatic malignant melanoma patients and to try to establish a correlation between the level of caspase-1 and different parameters related to this pathology. In addition, we evaluated the possible relationship between the clinical response to biochemotherapy and the caspase-1 level. The serum caspase-1 level was determined in 81 metastatic malignant melanoma patients and 50 normal volunteers using enzyme-linked immunosorbent assay (ELISA). Patients received cisplatin, recombinant interleukin-2 (Proleukin) and alpha-interferon (Roferon A) in two induction cycles, and assessment of clinical response was performed according to World Health Organization (WHO) criteria. The median caspase-1 level in melanoma patients was significantly higher (P = 0.0035) than in control samples. Interestingly, a positive correlation between caspase-1 level and the tumour burden was shown (rs = 0.629, P = 0.009). When the clinical response was taken into consideration, the level of caspase-1 was significantly higher in biochemorefractory patients compared with responding ones (P = 0.04). After treatment, the caspase-1 level remained very high in biochemorefractory patients, while in responding ones no change was observed. Furthermore, a positive correlation between the clinical response and the caspase-1 level was established (rs = 0.404, P = 0.024). In conclusion, we observed an elevated caspase-1 level in metastatic malignant melanoma patients. In addition, the correlations obtained between the caspase-1 level and both the tumour burden and the clinical response to the treatment support the concept that disrupted apoptosis pathways might be involved in the progressive disease of advanced melanoma and/or may confer resistance to treatment.

Plasma Fas ligand, an inducer of apoptosis, and plasma soluble Fas, an inhibitor of apoptosis, in advanced melanoma.

The transmembrane receptor Fas/APO-1, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death and induces apoptosis when it binds Fas ligand (FasL) or soluble Fas ligand (sFasL). However, soluble Fas (sFas) blocks apoptosis by inhibiting binding between Fas and FasL or sFasL. At present, the status of sFas and sFasL in metastatic malignant melanoma remains unknown. This study sought to evaluate the relationship between plasma levels of sFas and/or sFasL and clinical response in 45 metastatic malignant melanoma patients treated by biochemotherapy. sFas and sFasL were measured by specific enzyme-linked immunosorbent assay (ELISA) tests in the sera from patients and 34 healthy donors. Overall, sFas and sFasL levels in patients were significantly higher (P < 0.0001) than in healthy donors. Before the biochemotherapy treatment the sFas level was about the same in biochemorefractory (n = 26) as in responder patients (n = 19). In contrast, the sFasL level was very high only in biochemorefractory patients. At the end of the treatment, in biochemorefractory patients the sFas level was extremely significantly increased (P < 0.0001) and a significant decrease in the plasma levels of sFasL was observed (P = 0.0002). In responder patients, no change in sFas and sFasL was detected. In conclusion, elevated levels of sFas and sFasL might be associated with poor prognosis in advanced melanoma; their possible role in the regulation of apoptosis in influencing the response to biochemotherapy should be further explored.

A comparative study of Fas and Fas-ligand expression during melanoma progression.

BACKGROUND: Impaired regulation of apoptosis is known to be associated with the development of various cancers. Fas receptor (APO-1/CD95) binding to its ligand, Fas-ligand (Fas-L), has been shown to trigger apoptosis in various cell types. OBJECTIVES: In this study, we examined CD95 and Fas-L expression on primary and metastatic melanoma cells from patients to investigate a potential correlation between these measures of apoptosis and different disease stages. PATIENTS AND METHODS: Primary melanoma cells were obtained after surgical resection from 19 patients and metastatic cells from fine-needle aspiration of lymph nodes or palpable subcutaneous lesions in 25 patients. Normal skin cells were obtained at skin biopsy of 10 healthy donors. RESULTS: Flow cytometric analysis revealed that CD95 and Fas-L expression was detected in all the kinds of cell studied. In whole cell suspensions, CD95 expression was significantly higher (P < 0.0001) in normal skin cells than in melanoma cells, whatever the stage studied. By contrast, we observed an increase in Fas-L expression in melanoma cells compared with normal ones. Subsequently, using a double staining method, we studied these measures on HMB45+ cells, a specific marker for melanoma cells, and found that CD95 expression was significantly higher (P = 0.0005) in primary than in metastatic cells while Fas-L expression was significantly increased (P = 0. 0004) in metastatic compared with primary cells. Furthermore, a relationship was found between CD95 or Fas-L expression and Breslow thickness; as primary melanoma thickness progressively increased, the percentage of HMB45+ CD95+ cells decreased while that of HMB45+ Fas-L+ cells concurrently increased. CONCLUSIONS: These results suggest that downregulation of CD95 and upregulation of Fas-L in melanoma might be considered as concomitant with disease progression.

Effect of endogenous interleukin-6 on Fas (APO-1/CD95) receptor expression in advanced melanoma patients.

Interleukin-6 (IL-6) has been shown to support either autocrine or paracrine growth in melanoma, and may prevent programmed cell death in different cell types. We have previously demonstrated that the endogenous IL-6 level is significantly correlated with tumor burden and nonresponse to biochemotherapy in metastatic malignant melanoma patients. In the present study, we investigated the relationship between endogenous IL-6 and apoptosis signal through Fas (APO-1/CD95) receptor expression in 9 responder and 15 refractory patients with metastatic disease treated by biochemotherapy. Before any treatment, double immunostaining demonstrated that 61.5% of the tumor cells were HMB45+CD95+. At day 49 in refractory patients, a significant decrease (p = 0.04) of total Fas expression was observed. Furthermore, a significant reduction (p = 0.032) in the percentage of HMB45+CD95* cells occurred. An 11-fold increase in serum IL-6 level was detected (p < 0.002). This increase was negatively correlated (r = -0.2, p = 0.008) with the decrease in total Fas expression. However, in responding patients, no detectable decrease in Fas expression was observed, while a very low increase in serum IL-6 (2-fold) was detected. These results suggest that the increased endogenous IL-6 level in refractory patients may inhibit apoptosis via modulation of Fas expression. These preliminary results must be interpreted with caution, and further study with a greater number of patients is needed to understand the mechanism by which IL-6 inhibits apoptosis in melanoma.

Is there any relationship between interleukin-6/interleukin-6 receptor modulation and endogenous interleukin-6 release in metastatic malignant melanoma patients treated by biochemotherapy?

During recent years it has become clear that the production of most cytokines could play an important role in malignancies. We previously demonstrated that a high endogenous interleukin-6 (IL-6) level is significantly correlated with a high tumour burden and resistance to biochemotherapy in metastatic malignant melanoma patients. However, little is known about the origin of IL-6 and the pattern of IL-6 receptor (IL-6R) expression. In this report, we studied the expression of IL-6R and intracellular IL-6 using flow cytometry in tumour cells provided by fine-needle aspiration of lymph nodes and palpable metastatic lesions from 14 patients refractory to biochemotherapy and six responder patients. Moreover, we established the relationship between these parameters and the serum IL-6 level. Our results demonstrated that, following treatment, the percentage of HMB45-positive (HMB45+) cells expressing functional IL-6R, intracellular IL-6 or both IL-6R and IL-6 significantly decreased in patients refractory to biochemotherapy. In contrast, in responder patients the percentage of HMB45+ cells expressing IL-6R increased and those expressing IL-6 remained stable. Regarding the serum IL-6 level, an 11-fold increase was observed in the patients refractory to biochemotherapy, but only a 1.8-fold increase in the responder patients. In conclusion, in metastatic malignant melanoma patients with a poor prognosis, the endogenous production of IL-6 is concomitant with a decrease in functional IL-6R and intracellular IL-6 expression, suggesting the involvement of an IL-6/IL-6R complex.

Actions of HSVtk and connexin43 gene delivery on gap junctional communication and drug sensitization in hepatocellular carcinoma.

We have previously demonstrated that transfected hepatocellular carcinoma cells (Hepa1-6) with one copy (pAGO) and two copies (pYED) of the HSVtk gene, using liposomes, induced cell death of untransfected cells in the presence of ganciclovir (GCV). This phenomenon is called the 'bystander effect'. To determine whether an elevated level of connexin43 increases the bystander effect, we have cotransfected Hepa1-6 cells with a plasmid containing the HSVtk gene driven by the alpha-fetoprotein promoter (pFTK) or pAGO or pYED and connexin43. The results showed that, after GCV treatment, the percentage of growth inhibition was higher (25-30%) in cells cotransfected with HSVtk and connexin43 than in cells transfected only with HSVtk gene. The IC50 of GCV on cells transfected with pFTK/Connexin43 was 17.85-fold lower than cells transfected with pFTK alone. To improve these results, stable connexin43 transduced Hepa1-6 cells were transfected with pFTK followed by GCV treatment. In this case, the cell growth was markedly inhibited as compared with parental cells. Furthermore, we have studied the correlation between the expression of the HSVtk and the connexin43 proteins. Using flow cytometric analysis, scrape loading/dye transfer and immunoblotting assay we found that the cells transfected separately by pAGO, pYED, pFTK and pLTR-Cx43 showed an increase of connexin43 protein. This study indicates that transfecting Hepa1-6 cells with both connexin43 and HSVtk genes up-regulates connexin43 expression which enhances the bystander effect and subsequently tumor cell death.

Cytoskeleton alteration in MCF7R cells, a multidrug resistant human breast cancer cell line.

Various cytoskeleton modifications are associated with malignant cell transformation and have been used as prognostic factors. A human breast cancer cell line (MCF7S) and its multidrug resistant (MDR) subline (MCF7R) were characterized here for their intermediate filaments (IFs) expression (cytokeratin 8, 18, 19 and vimentin) as a function of their resistance phenotype. Modifications of these cytoskeleton molecules were analyzed by flow cytometry, immunofluorescence, electrophoresis and immunoblotting techniques. Cytokeratins 8 and 18 were similarly expressed in the cell lines. Cytokeratin 19 was expressed in the MCF7S cell line and not in the MCF7R variant, while vimentin was highly expressed in MCF7R and slightly in MCF7S. Analysis of IFs after the addition of doxorubicin (Dox) in the culture medium of MCF7S, showed an increase in cytokeratin 8 filaments. Vimentin expression in MCF7R was not modified in the presence of these different MDR modulators. Acquisition of MDR was associated with an increase and a redistribution of vimentin filaments characterized by a perinuclear polarization. These drug resistance associated changes might derive from different biological processes triggered by chemotherapy. In conclusion, this suggests that this intermediate filament could be a marker associated with chemoresistance or a marker of malignancy in certain epithelial cancers.

A flow cytometric method for the rapid detection of beta-galactosidase transfected cells: an in vitro and in vivo study.

A flow cytometric method has been developed for the rapid analysis of lacZ transduced cells. The method described is based on an indirect immunofluorescence staining procedure using a monoclonal antibody which binds specifically to beta-galactosidase from E. coli and to beta-galactosidase fusion proteins. This technique was used for the quantification in vitro as well as in vivo of beta-galactosidase expression in B16 melanoma cells. The described method is appropriate for a variety of cell types (species, lineage), is simple, quantitative, reliable, rapid and applicable to all constructs containing the lacZ selectable markers. It should prove to be very helpful (1) for the quantification of cells expressing the lacZ reporter gene and (2) for studying gene regulation, including transfection modality, promoter efficacy, enhancer activity, and other regulatory factors.

Liposomal delivery of the herpes simplex virus thymidine kinase gene in glioma: improvement of cell sensitization to ganciclovir.

In this study, we investigated whether the regulation and the copy number of the herpes simplex virus thymidine kinase (HSVtk) gene increased the sensitization to ganciclovir (GCV) of glioma cell lines (Rat C6 and human U118-MG) using liposome-mediated gene transfer. Three recombinant plasmids carrying the HSVtk gene driven by the thymidine kinase promoter in single (pAGo) and double copy (pYED) or by the human cytomegalovirus promoter (pCMVtk) were used for the transfection. The DNA delivery was optimized by screening a panel of cationic liposomes using Lac-Z and luciferase as reporter genes. The efficiency of transfection reached 33% to 36% in vitro but only 18.6% in vivo after an intratumoral injection of DNA-liposome complexes. Moreover, after transfection of the three plasmids, the cell-killing effect of GCV was evaluated. A significant enhancement (four- to fivefold) of the cell sensitivity to GCV was shown in pCMVtk and pYED as compared with pAGo-transfected cells in both cell lines. According to the plasmid, the effect of the HSVtk/GCV system was confirmed by in vivo experiments and was objectified by a higher tumor weight reduction with pCMVtk (49%) than pAGo (27%). From these results, we conclude that (1) the gene transfer can be achieved by cationic liposomes both in vitro and in vivo and that (2) using this type of vector, the antitumor effect of the HSVtk/GCV system could be potentiated by the up-regulation of HSVtk gene duplication.

In vitro sensitization of the B16 murine melanoma cells to ganciclovir by different RNA and plasmid DNA constructions encoding HSVtk.

The antiviral drug ganciclovir (GCV) is toxic for mammalian cells transfected with the herpes simplex virus thymidine kinase (HSVtk) gene. To improve the results obtained by our group previously on nonviral transfection of tumor cells, we have examined here in vitro virus-free transfection of murine B16 melanoma cells via lipofectamine-nucleic acid complexes carrying either HSVtk gene transcripts or plasmid DNAs containing single and double copies of the HSVtk gene. The HSVtk gene transcripts as well as plasmids containing the HSVtk gene(s) rendered cells sensitive to GCV treatment. Tumor sensitivity to GCV conferred by the HSVtk gene transcripts was of the same level as the sensitivity conferred by plasmid DNAs containing a single copy of the HSVtk gene. However, when the plasmids containing double copies of the HSVtk gene were used, sensitivity to low GCV concentrations increased dramatically. One could appreciate this finding as an essential advantage of the plasmids containing double copies of the HSVtk gene since it allows use of the GCV concentration range which is common in clinical applications.

Endogenous interleukin 6 levels in patients with metastatic malignant melanoma: correlation with tumor burden.

The involvement of interleukin (IL-) 6 in malignant disease has been investigated in a variety of different malignancies. To evaluate whether serum IL-6 is a useful disease marker in metastatic malignant melanoma (MMM), we studied the time course of endogenous IL-6 secretion in 41 patients treated with cisplatinum, IL-2, and IFN-alpha. Furthermore, the relationship of endogenous IL-6 concentrations to the tumor burden and/or the clinical response was also evaluated. The baseline serum IL-6 levels were significantly higher in patients with MMM than in the control group (P = 0.002). When tumor burden was taken into consideration, we found that IL-6 levels were higher in patients with high tumor burden than in patients with low tumor burden. During treatment in the whole patient population, a higher serum IL-6 level was observed in nonresponding as compared to responding patients at days 7 (P = 0.0005), 21 (P = 0.002), and 35 (P = 0.009). The follow-up of serum IL-6 in patients with MMM according to the tumor burden and clinical response demonstrated that: (a) IL-6 levels were significantly higher at days 7 and 21 in patients with high tumor burden as compared to those with low tumor burden; and (b) IL-6 levels remain significantly higher in nonresponding patients as compared to responding patients regardless of the tumor burden. From these results, we can conclude that endogenous IL-6 may play a role in the failure of IL-2 therapy in such patients, since the very early IL-6 increase is correlated with the tumor mass and nonresponse to biochemotherapy. Therefore, it seems that the early detection of endogenous IL-6 may represent valuable information for monitoring the response to biochemotherapy in patients with MMM.

Gene transfer in hepatocarcinoma cell lines: in vitro optimization of a virus-free system.

Many approaches exist for hepatic gene delivery, including viral vectors and non-viral vectors. In this study, we tested a panel of liposomes to transfer pAGO, a plasmid containing one copy of herpes simplex virus (HSVtk) gene, and pYED11, a plasmid containing two copies of the HSVtk gene, into a murine hepatocarcinoma cell line (Hepa 1-6) and a human hepatocarcinoma cell line (Hep-G2). The efficiency of gene delivery and expression was characterized by beta-galactosidase staining, flow cytometric analysis and quantitative lacZ activity. Different combinations of liposomes and DNA and the ratio of the concentration of liposome to DNA were tested. The efficient transfer was shown with DOTAP followed by transfectam and lipofectamine. Under these conditions, we tested the cytotoxicity of ganciclovir (GCV) exposure on Hepa 1-6 and Hep-G2 transfected separately with liposome-pAGO and liposome-pYED11 complexes. This study demonstrates the in vitro efficacy of each liposome tested to transduce the HSVtk gene into hepatocarcinoma cell lines. The transfer of two copies of the HSVtk gene rendered cells 1.5 times more sensitive to GCV than cells transduced by pAGO as compared to controls. This was achieved most efficiently by the DOTAP-pYED11 complex. Thus, pYED11 may be considered as an alternative to pAGO as a gene transfer vector.

Direct gene transfer of a plasmid carrying the herpes simplex virus-thymidine kinase gene (HSV-TK) in transplanted murine melanoma: in vivo study.

The aim of the study was to use a virus-free system to transfer the Herpes Simplex Virus-thymidine kinase (HSV-TK) gene in mice bearing melanoma tumours. B16 F1 murine melanoma cells were injected subcutaneously. On days 11 and 14, an intratumoral injection of either naked plasmid containing the HSV-TK gene (pAG0) or pAG0-lipofectamine complexes was given. Ganciclovir (120 mg/kg/day) was given for 5 days starting on day 14. Tumour weight reduction (40-50%) was observed in treated animals versus different control groups. Moreover, histopathological analysis on tumours showed large areas of cavitary necrosis (85%) in treated groups compared to controls (10%). Using a simple and safe method, the results presented here demonstrated that virus-free mediated delivery of the HSV-TK gene is efficient in vivo in murine malignant melanoma.

Virus-free transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir treatment induces tumor cell death.

We report virus-free transfer of a "suicide" gene into tumoral cells. The system can be used in vitro or in vivo to induce tumor cell death. A plasmid carrying the herpes simplex virus thymidine kinase (HSV-TK) gene with its 5'- and 3'-flanking regions was used both alone and in liposomes to transduce B16 cells. In vitro, a 5-day treatment with ganciclovir after transfection with the HSV-TK gene in liposomes induced a significant lysis of B16 melanoma cells as assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The efficacy of transfection was determined using liposomes harboring the beta-galactosidase reporter gene and was around 10%. Thus, the cytotoxicity observed resulted presumably from a large bystander effect. In vivo, direct transfer of the TK DNA into established B16 melanoma tumors in C57B6 mice followed by i.p. ganciclovir treatment induced a 50% reduction of tumor weight after 8 days and an increased necrosis. Despite the use of the nonspecific strong TK promoter, no necrosis was detected in normal tissues surrounding the tumor or elsewhere. Thus, this system of tumor transfection, which does not involve any viral vector, is safe and straightforward and seems to be suitable for testing in clinical trials.

Comparison of the uptake of retinoids 13-cis-retinoic acid and Ro 13-6298 delivered to HL-60 cells by serum albumin or low-density lipoprotein.

Retinoids, a class of polyisoprenoids including retinol and retinoic acid, regulate and control diverse physiological functions via their cell-differentiating and morphogenic potential. In the present study we showed that the extracellular concentration of retinoid-binding proteins such as albumin limits the amount of retinoid entering the human promyelocytic leukemia cell line HL-60. These cells accumulate 5 -10 times more retinoid when delivered free in solution than when bound to either albumin or low-density lipoprotein (LDL). Moreover. the effect of protein binding is concentration-dependent, with a higher concentration of binding protein corresponding to a lower level of cellular uptake. Furthermore, the uptake of the ester derivative is higher than that of the acidic retinoid. These observations suggest that (a) the cellular uptake of both retinoids occurs via the free form of the ligand in solution, with the free concentration of ligand decreasing as the carrier-protein concentration increases, and (b) according to a passive mechanism, the ester derivative, unionized and lipophilic, enters the cells more easily than does the acidic derivative.

Study of IL-2 receptor expression after chemoimmunotherapy in patients treated for metastatic malignant melanoma.

Using flow cytometry, cellular IL-2 receptors were studied before and following chemoimmunotherapy combination in 20 patients with metastatic malignant melanoma (MMM). Patients received cisplatin (100 mg/m2) at days 1 and 28, recombinant IL-2 by continuous infusion from days 3 to 6, 17 to 21, 31 to 34, and 45 to 49. Interferon-alpha (IFN-alpha) was given subcutaneously three times weekly. In terms of clinical response, we observed 55% objective response (complete: 15%). When pretreatment blood samples were compared with those of healthy donors, we did not observe any change in low (alpha chain) and high affinity receptor (alpha + beta) expression. In contrast, intermediate affinity p75 (beta chain) expression was decreased significantly (P < or = 0.0001) in MMM patients. During treatment, we found a dramatic increase of beta chain as well as high affinity (alpha + beta) expression in responding patients, as soon as IL-2 therapy began. Furthermore, the increase of beta chain expression was limited to natural killer (NK) cells (CD56+). In non-responding patients, on the other hand, increase of both receptors was seen only at day 31. These data suggest the involvement of beta chain expression in the mechanism of cell activation after chemoimmunotherapy. Moreover, this early beta chain expression is correlated with the clinical response to chemoimmunotherapy.