人急性早幼粒细胞性白血病细胞诱导分化生成树突状细胞的体外研究

树突状细胞(dendritic cells,DC)在激发机体抗白血病T细胞免疫应答中具有重要作用,有报道DC可以由单核细胞及粒细胞分化生成,本研究应用细胞因子在体外诱导了急性早幼粒细胞性白血病(APL)细胞向DC的分化.从M3型APL患者外周血分离白血病细胞,加入GM-CSF(100ng/ml)或GM-CSF(100ng/ml) rhIL-4(500U/ml)体外培养14天,并于培养结束前3天加人TN-α(100ng/ml).结果表明,GM-CSF可以促进白血病细胞体外增殖,并从幼稚状态逐渐分化成熟,表达高水平的CD45分子,其中部分为CD14~ 的单核细胞,部分为CDla~ 的DC(M3DC);培养后期加入TNF-α,可以促进DC生成,约占35%;以GM-CSF IL-4培养也诱导了幼稚细胞的成熟,2周后DC约占10%,但较少单核细胞生成,培养至3周时DC约占60%;而在培养后第11天加入TNF-α则可以加速DC生成,3天后生成的白血病型DC达90%.电镜观察到M3DC具有与单核细胞来源的DC相似的超微结构,但具有的突出特征是部分细胞存在少量胞浆颗粒;M3DC高表达HLA-DR、B7-2、CD40、CD54分子,体外可以强烈刺激同种T细胞增殖.此类由白血病细胞诱导生成的DC可被用于体内外诱导生成肿瘤特异性CTL,在APL的免疫治疗中具有一定的应用价值.     

Antitumor Effect of Recombinant Human Interleukin-1β Alone and in Combination with Natural Human Tumor Necrosis Factor-α

In order to investigate the antitumor effect of recombinant human interleukin-1β (IL-1β) alone and in combination with natural human tumor necrosis factor-α (nHuTNF-α), we used female BDF₁ mice bearing Lewis lung carcinoma (3LL). IL-1β showed an antiproliferative effect against pulmonary metastatic tumors of 3LL in a dose-dependent manner. We observed 19.6 ± 6.6, 18.6 ± 5.3, 14.1 ± 4.4 and 13.0 ± 6.0 metastatic tumors at doses of 0.5, 1.0, 2.5 and 5.0 μg IL-1β/mouse/day by daily intravenous administration (the number of metastatic tumors of the control group was 26.3 ± 8.2). Similar results were obtained by intraperitoneal administration, but in this case, mice showed a marked decrease of body weight. When IL-1β was administered in combination with nHuTNF-α, pulmonary metastatic tumors decreased much more than when IL-1β was administered alone. When the control group had 18.6 ± 12.7 metastatic tumors, the nHuTNF-α group had 12.3 ± 3.9 and the IL-1β group had 12.8 ± 8.0, the group which was administered both cytokines had a significantly decreased number of 5.6 ± 3.3 metastatic tumors. This antiproliferative effect of IL-1β in combination with nHuTNF-α was reduced by the intravenous administration of anti-asialo GM₁ antibody and carrageenan. The number of metastatic tumors was increased from 8.9 ± 8.0 to 18.8 ± 11.4 by anti-asialo GM₁ antibody and from 9.5 ± 6.8 to 28.0 ± 12.3 by carrageenan. It was suggested that asialo GM₁-positive cells and macrophage were two of the most important effectors of the antiproliferative effect of IL-1β and TNF-α.     

γδ T Cells Predict Outcome in Zoledronate‐Treated Breast Cancer Patients

The biological mechanism underlying the antitumor role of zoledronate is unclear. The analysis in this letter illustrates the diagnostic and prognostic potential of a γδ T-cell-based blood test and implies a link between immune responsiveness and positive outcome with zoledronate therapy.We read with great interest the article by Valachis et al. [1], published in The Oncologist. In a comprehensive meta-analysis of 15 randomized clinical trials on adjuvant therapy for breast cancer patients with zoledronate, the authors identified a significant overall survival benefit with zoledronate treatment, in agreement with one smaller meta-analysis [2] but not another [3] conducted earlier. These new findings support the call for zoledronate to be considered as a new standard of care in adjuvant breast cancer therapy [4].The dilemma remains that the biological mechanism underlying the antitumor role of zoledronate is unclear, as Valachis et al. correctly highlight [1]. The clinical benefit may in fact stem, at least in part, from the activity of zoledronate on the patient''s immune system by specifically stimulating Vγ9/Vδ2 T cells, which compose 0.5%–5% of circulating T cells in healthy adults [5]. Targeted immunotherapy studies directly exploiting the potent non-major histocompatibility complex-restricted cytotoxicity of Vγ9/Vδ2 T cells have shown excellent safety profiles and promising clinical observations in a variety of hematological and solid cancers [6], including metastatic breast cancer [7].Studies by us and others have demonstrated that zoledronate treatment induces a rapid and long-lasting conversion of the peripheral Vγ9/Vδ2 T-cell phenotype from CD27⁺CD45RA⁻ central memory T (T_CM) cells to CD27⁻CD45RA⁻ effector memory T (T_EM) cells in many, but not all, individuals [7–9]. In Figure 1, zoledronate-treated breast cancer patients with stable disease showed elevated proportions of Vγ9/Vδ2 T_EM cells at the time of blood sampling compared with patients with progressing disease and untreated controls (Fig. 1A). No such differences were observed with regard to patient age, time on treatment, or pretreatment clinical status. We then stratified the same patient cohort according to their follow-up disease status at 3–9 months after blood sampling. This showed successful conversion from T_CM to T_EM cells (as evident by a lower frequency of T_CM cells) in patients who were stable 3–9 months after blood sampling compared with patients who went on to show progressing disease (Fig. 1B).Open in a separate windowFigure 1.γδ T-cell memory subsets in metastatic breast cancer patients in relation to clinical outcome. (A): Proportion of CD27⁻CD45RA⁻ effector memory T cells among circulating Vγ9/Vδ2 T cells in 10 zoledronate-treated patients in relation to clinical outcome at the time of sampling and in comparison to three untreated patients. (B): Proportion of CD27⁺CD45RA⁻ central memory T cells in relation to subsequent clinical outcome at 3–9 months after sampling. Patients received at least two cycles of zoledronic acid (Zometa; Novartis International, Basel, Switzerland, http://www.novartis.com) for >1 month and <8 months and had no other active malignancies within the previous year; no active or uncontrolled infections; no autoimmune disorders or serious allergic reactions; no use of immunosuppressives during the previous 3 months; and no prior chemotherapy or cytotoxic agents during the previous 6 months.Abbreviations: AUROC, area under the receiver operator curve; CI, confidence interval; PD, progressing disease; SD, stable disease; T_CM cells, central memory T cells; T_EM cells, effector memory T cells.Our analysis illustrates the diagnostic and prognostic potential of a γδ T-cell-based blood test and implies a link between immune responsiveness and positive outcome with zoledronate therapy, thereby contributing to our understanding of the as yet unexplained clinical benefit of adjuvant therapy. These findings are particularly intriguing because new treatments specifically targeting bone resorption, such as denosumab, lack this potential to act on γδ T cells and may not yield the direct antitumor benefit observed with zoledronate.     

Curcumin-induced Apoptosis in HL-60 Human Leukemic Cells

Curcumin is the main biologically active phytochemical compound in turmeric. It has been shown to have ‍anticarcinogenic activity. The aims of the study were to identify the mechanism of apoptosis of HL-60 human ‍promyelocytic leukemic cells induced by curcumin and to determine the effects of water-soluble antioxidants, ascorbic ‍acid, Trolox (a water-soluble form of vitamin E), glutathione (GSH) and N-acetylcysteine (NAC) on this process. ‍HL-60 cells were incubated with curcumin for 24 h and apoptotic cells were quantitated by flow cytometry following ‍staining with annexin V-FITC and propidium iodide. Curcumin-treated HL-60 cells produced reactive oxygen ‍species as detected by the dichlorofluorescein fluorescent assay. Apoptosis occurred via the mitochondria pathway ‍as curcumin reduced mitochondrial membrane potential in a dose-dependent manner. In the presence of 10 iM ‍curcumin, vitamin C (56 nM – 5.6 iM) inhibited apoptosis of HL-60 cells; GSH at low concentration (1 iM) reduced ‍apoptosis but had no effect at higher concentrations (10, 100 iM); and Trolox and NAC at 10 and 100 iM, respectively, ‍enhanced apoptosis, but this effect was abolished at higher concentration (1 mM) of NAC. MAPKK/MEK inhibitor ‍PD98059, enhanced curcumin-induced HL-60 apoptotic cell death.     

Phenotypic characterization and prognostic impact of circulating γδ and αβ T‐cells in metastatic malignant melanoma

Human T cells carrying γδ T‐cell receptors (TCRs) represent a minor population relative to those with αβ TCRs. There has been much interest recently in the possibility of using these γδ T‐cells in cancer therapy because they can kill tumor cells in vitro in an MHC‐unrestricted manner, and possess potential regulatory capability and antigen‐presenting capacity. The presence of γδ T‐cells in late‐stage melanoma patients and their relationship with survival has not been extensively explored, although relatively lower percentages of total γδ T‐cells and Vδ2+ cells have been reported. Here, we present a detailed analysis of associations of γδ T‐cell subsets and differentiation stages with survival in Stage IV patients, compared with CD4+ and CD8+ αβ T‐cells. We found an increased Vδ1:Vδ2‐ratio and a decreased CD4:CD8‐ratio in patients compared to healthy controls, on the basis both of relative frequencies and absolute cell counts per μL blood. Nonetheless, Kaplan–Meier analyses showed that a higher than median frequency of Vδ1+ cells was negatively associated with survival, whereas there were no positive or negative associations with frequencies of Vδ2+ cells. Correlations of cell differentiation status with survival revealed a negative association of early‐differentiated Vδ1+ T cells with survival, both on the basis of relative frequencies and absolute counts. There was also a positive correlation between the frequencies of early‐differentiated CD8+ αβ T‐cells and survival. Our findings suggest peripheral blood frequencies of Vδ1+ T‐cells as a potential prognostic marker in melanoma. The mechanisms by which higher abundance of Vδ1+ cells are associated with poorer survival require determination.     

Secretion of Transforming Growth Factor-ß by Human Myelogenous Leukemic Cells and Its Possible Role in Proliferation of the Leukemic Cells

Transforming growth factor(TGF)-ß activity was found in the neutral extracts of human myelogenous leukemic cells or K562 cells and the conditioned medium from K562 cell culture. BALB/c 3T3 cells grown in soft agar in the presence of TGF-ß₁ produced an activity that stimulated the growth of K562 cells. This activity was non-dialyzable. acid-stable, heat-sensitive and partially Inactivated by pronase treatment. These results suggest a mutual growth reliance between the leukemic cells and fibroblasts mediated by paracrine growth factors produced by these cells.     

Expression of phospholipases γ1, β1, and δ1 in primary human colon carcinomas and colon carcinoma cell lines

The levels of expression of phosphoinositide-specific phospholipase Cs (PLCs) were examined in a series of primary human colon carcinomas and in eight colon carcinoma cell lines by using monoclonal antibodies and cDNA probes for PLCγ1, PLCβ1, and PLCδ1. Western and northern blot analyses of PLCγ1 revealed elevated expression of this isozyme at both the protein and mRNA levels in most tumors when compared with paired adjacent normal mucosa samples (in 11 of 13 pairs in the western blots and 8 of 9 pairs in the northern blots). On the other hand, decreased levels of the PLCδ1 protein were seen in most colon carcinomas (12 of 13 paired samples). The levels of PLCβ1 protein were too low to detect possible differences between the carcinoma and normal mucosa samples. Relatively high expression of PLCγ1 was found in almost all of the eight human colon carcinoma cell lines at both the protein and mRNA levels. Only weak expression of PLCβ1 was detected in these cell lines, by both western and northern blot analyses, and PLCδ1 protein was not detected in any of the carcinoma cell lines. These findings provide evidence that colon carcinomas display altered expression of individual isoforms of PLCs and suggest that increased expression of PLCγ1 may play an important role in colon carcinogenesis. © 1995 Wiley-Liss Inc.     

Estrogen receptor α and β in esophageal squamous cell carcinoma

A gender difference has been reported in the morbidity of esophageal squamous cell carcinoma (ESCC). Estrogens have been proposed to play a role in this difference but the details have not yet been clarified. Therefore, in the present study, we examined the status of estrogen receptor (ER)α and ERβ in 90 Japanese ESCC patients. ERα and ERβ immunoreactivity was detected in the nuclei of ESCC cells (41.1 and 97.8%, respectively). There was a significant positive association between the ERβ H score and histological differentiation (P = 0.0403), TNM‐pM (LYM) (P = 0.00164) and Ki67/MIB1 LI of carcinoma cells (P = 0.0497, r = 0.207). In addition, the ERβ status of carcinoma cells was significantly correlated with unfavorable clinical outcome of the patients. Multivariate analysis further revealed the ERβ status in carcinoma cells as an independent unfavorable prognostic factor of these patients. We further examined the effects of estrogen treatment on ESCC cell line (ECGI‐10) transfected with ERα or ERβ in vitro. The number of ECGI‐10 transfected with ERβ was increased by estradiol or ERβ specific agonist but estradiol did not exert any effect upon the cell number of ECGI‐10 transfected with ERα. In summary, the results of the present study clearly demonstrate that the status of ERβ in ESCC was closely associated with the unfavorable prognosis, possibly through altering cell proliferation of carcinoma cells. (Cancer Sci 2012; 103: 1348–1355)     

Tumorigenicity of IL-1α- and IL-1β-Deficient Fibrosarcoma Cells

Analyzing the growth of fibrosarcoma lines derived from IL-1α-, IL-1β- , or IL-1αβ-knockout (^-/-) mice in the immunocompetent host revealed that tumor-derived IL-1α and IL-1β exert strong and opposing effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1-deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1-deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1α^-/-β-competent (^comp) lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1β^comp lines, IL-1α strengthens anchorage-independent growth, but both IL-1α and IL-1β support drug resistance. Corresponding to the aggressive growth, IL-1β^comp cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated α_v/β₃ and matrix metalloroteinase expression. Recruitment of myeloid cells requires IL-1β but is regulated by IL-1α, because inflammatory chemokine and cytokine expression is stronger in IL-1α^-/-β^comp than in IL-1^wt lines. This regulatory effect of tumor-derived IL-1α is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1β. Both sarcoma cell-derived IL-1α and IL-1β promote tumor growth. However, IL-1α exerts regulatory activity on the tumor cell-matrix cross-talk, and only IL-1β initiates systemic inflammation.     

Expression of β1 integrin receptors in transformed mouse epidermal keratinocytes: Upregulation of α5β1 in spindle carcinoma cells

The adhesive properties and the expression of extracellular matrix receptors of the β1-integrin subfamily were analyzed in transformed epidermal keratinocyte cell lines of different stages of mouse skin carcinogenesis. One- and two-dimensional analyses of the immunoprecipitates obtained with anti-β1- and specific anti-α-integrin subunits showed qualitative and quantitative changes in the expression of β1 integrins by the different cell lines. The polyvalent α3β1 integrin was expressed by all analyzed cell lines, although the levels detected in undifferentiated spindle CarC cells were lower than those present in the rest of keratinocyte cell lines. In contrast, spindle cells expressed high levels of the specific fibronectin receptor α5β1, whereas this integrin was absent or expressed at very reduced levels in the other epithelial cell lines. Expression of α5β1 integrin in spindle cells appeared organized in cell-substratum contact areas on spread cells. In addition, high and homogenous expression of α5β1 was detected in fully undifferentiated tumors induced in nude mice by three independent spindle cell lines. These results suggest that the expression of α5β1 integrin is upregulated during the development of spindle cell carcinomas that occur in the last stages of mouse skin carcinogenesis and can be associated with the acquisition of the fibroblastoid phenotype of spindle cells. On the other hand, expression of the collagen receptor α2β1 was demonstrated in a transformed cell line (PDV), and it was apparently also expressed in two other malignant keratinocyte cell lines (PDVC57 and HaCa4). The expression of α2β1 was correlated with the increased adhesion to collagen type I and Collagen type IV exhibited by the tumorigenic cell lines. © 1995 Wiley-Liss Inc.     

Expression of α3β1 integrin receptor and its ligands in human lung tumors

Increasing experimental evidence demonstrates that malignant transformation is associated with changes in the repertoire of expression of the integrin family of molecules, which mediate cell-matrix and cell-cell interactions. We have analyzed immuno-histochemically and immunochemically the expression of VLA-3 integrin and its known ligands, namely, laminin (LM), fibronectin (FN), collagen type IV (Coll IV), nicein (NIC), and entactin/nidogen (ENT), in lung tumors of various histological types. α₃β₁ was detectable in normal bronchial epithelium and along basement membranes of alveolar walls. In non-small cell lung carcinomas (NSCLC) the integrin was expressed in 82% of the cases, independently of histological type and degree of differentiation of the tumors. On the other hand, only 13% of the small cell lung carcinomas (SCLC) displayed a weak and heterogeneous distribution of the α₃β₁ complex. Our findings were confirmed immunochemically using long-term tumor cell lines. While the expression of both α₃β₁ and ligands LM, FN, Coll IV, and Ent correlated in NSCLC with the presence of basement membranes, FN was the only ligand detectable in the stroma of SCLCs. A selective loss of nicein in basement membranes was demonstrated in NSCLC indicating an impairment of expression of this glycoprotein following malignant transformation. © 1995 Wiley-Liss, Inc.     

Polarization of the α6β4 integrin in ovarian carcinomas

By immunizing a BALB/c mouse with a human ovary-carcinoma cell line (IGROVI), grown intraperitoneally in nude mice, a monoclonal antibody (MAb), designated MAR6, was produced and characterized. Immunofluorescence on the immunizing cell fine showed a specific labelling by MAR6 at the cell-to-cell contact points. In addition, MAR6 was found to immunoprecipitate the α6β4 integrin complex. Competition tests with MAbs anti-α6, anti-β4, anti-β1 sub-units demonstrated that the recognized sub-unit is α6. Indirect immunofluorescence on various cell lines gave MAR6 as positive only on α6-positive lines (IGROVI, OVCAR3, SW626, SKOV3, ME4405, Calu3, N592, MDA468, A431 and HT29). Moreover, on IGROVI and OVCAR3 ovary-carcinoma cells, which normally grow either adhering to the culture flask or forming clumps in suspension in the medium, MAR6 selectively stained the connection points between the cells in clumps, where, in the same position, the presence of the β4 sub-unit, laminin and fibronectin was detected. On the contrary, the β1 sub-unit was distributed over the whole cell membrane. The same pattern of labelling by these MAbs was observed in 2 cases of ovarian-carcinoma cells present in ascitic fluids obtained from patients. Immunoperoxidase tests performed on cryosections of various normal tissues showed specific reactivity of MAR6 on basal or basolateral membranes of epithelial cells. On cryosections of ovarian tumors, MAR6 reactivity correlated with the degree of tumor differentiation. Indeed, in benign and well-differentiated tumors, a strong basal or basolateral labelling only of cells surrounding the neoplastic nodules was found. On the contrary, on undifferentiated tumors the inner part of the tumor nodules was also progressively labelled, whereas the staining on the border was weak and discontinuous as a result of the α6 sub-unit dispersion on the tumor cell surface.     

Interleukin-1α gene expression and localization of interleukin-1α protein during tumor promotion

Treatment of the dorsal epidermis of SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and dose-dependent stimulation of interleukin-1α (IL-1α) gene expression. Levels of IL-1α mRNA were elevated as early as 15 min and peaked at 3–4 h after a single application of TPA (2 μg or 10 μg). IL-1α gene expression increased in epidermal tissue isolated from SENCAR mice at 1, 3, 6, 10, 16, and 22 wk after a single treatment with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent twice-weekly application of 2 μg TPA. IL-1α-immunoreactive protein was specifically localized within suprabasal keratinocytes in cutaneous tissue isolated from mice treated with DMBA-TPA for 1–22 wk and in nonproliferating cells located within papilloma tissue isolated from SENCAR mice at 22 wk after initiation and promotion. Basal cells within hyperplastic epidermis did not produce IL-1α-immunoreactive protein. DMBA treatment alone did not induce IL-1α gene expression. Injection of IL-1α-specific antibodies (50 μg) into SENCAR mice via the tail vein 2 h before treatment with TPA (2 μg or 10 μg) significantly (P < 0.05) inhibited the skin thickening usually observed 24 h after treatment with TPA. Autoradiography studies showed that injection of anti—IL-1α antibodies inhibited incorporation of [methyl-³H]thymidine by keratinocytes within the epidermis and by cells within hair follicles. It also inhibited neutrophil infiltration into the dermis, which usually results from topical application of TPA. These data suggest that IL-1α is a pivotal cytokine produced by specific subpopulations of epidermal keratinocytes and that IL-1α primarily regulates the epidermal proliferative response of a distinctly separate population of keratinocytes after topical exposure of murine epidermis to TPA and secondarily modulates neutrophil migration into the dermis. Consequently, manipulation of IL-1α may be a way to attenuate or abrogate the cutaneous response to TPA by altering keratinocyte proliferation, the resultant hyperplasia, and a portion of the inflammatory response characterized by dermal infiltration of neutrophils.     

Analysis of mdr-1 Gene Expression in Human Leukemic Cells by Quantitative Competitive PCR

The ability to recognize the acquisition of multidrug resistance (MDR) in leukemia patients would improve our ability to predict the responsiveness of patients to chemotherapy. To quantitate the degree of MDR acquisition, we determined the amount of mdr-1 mRNA in leukemic cells from patients by competitive polymerase chain reaction (PCR) analysis. Twenty-one patients including 12 patients prior to treatment and nine relapsed patients with acute myelogenous or lymphoblastic leukemia were examined. The amount of mdr-1 gene expression in K562, K562/ADR₅₀₀ cells and their mixtures showed a proportional correlation between the ratio of resistant to non-resistant cells and the amount of the mdr-1 gene. Mean mdr-1 gene expression in relapsed patients was greater than that in pretreatment patients. Patients refractory to chemotherapy (NR) showed higher levels of mdr-1 gene expression than the patients who achieved complete remission (CR). Because of the wide variations in values, no statistical differences were observed between pretreatment and relapsed patients, or CR and NR patients. These results suggest that the competitive PCR technique is a reliable method to quantitatively determine mdr-1 gene expression, but it may be difficult to predict responsiveness to chemotherapy by using this technique alone.     

αv-Integrins in human melanoma: Gain of αvβ3 and loss OF αvβ5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice

We investigated the expression of α_v-integrins in different stages of human cutaneous melanocytic tumor progression. We observed that α_vβ₅ was the α_v-integrin expressed in all common nevocellular nevi, in 78% of dysplastic nevi, in 63% of early primary melanomas, in 43% of advanced primary melanomas, and in 33% of melanoma metastases. Hence, loss of α_vβ₅ expression was related to melanocytic tumor progression. In line with earlier reports, α_vβ₃ was exclusively detected in advanced primary melanomas and metastases (24% and 50% respectively). Staining with anti-α_v monoclonal antibodies (MAbs) in lesions where both α_vβ₃ and α_vβ₅ were absent showed that alternative α_v-integrins were expressed in advanced primary melanomas and metastases. By FACS analysis, we determined expression of α_vβ₅ and α_vβ₃ in 4 human melanoma cell lines with different metastatic capacities after s.c. inoculation into nude mice. One of the non-metastatic and both highly metastatic cell lines expressed α_vβ₅ at their surface. Surprisingly, α_vβ₃ was detected exclusively in the non-metastatic cell lines. Absence of α_vβ₃ in the highly metastatic cell lines was confirmed by lack of immunoprecipitation from ³⁵S-methionine-labeled cells and by absence of immunohistochemical staining on primary and metastatic xenograft lesions. Our findings indicate that α_vβ₅ expression is often lost in advanced stages of melanocytic tumor progression in situ, while α_vβ₃ is acquired, but that a decrease in α_vβ₅ and an increase in α_vβ₃ expression are not necessarily related to the metastatic behavior of human melanoma cells in nude mice. © 1995 Wiley-Liss, Inc.     

Cell Density-Dependent Proliferative Effects of Transforming Growth Factor (TGF)-β1, β2, and β3 in Human Chondrosarcoma Cells HCS-2/8 Are Associated with Changes in the Expression of TGF-β Receptor Type I

In this study, the growth properties of the human chondrosarcoma cell line HCS- 2/8, its response to transforming growth factor (TGF)-β isoforms 1, 2, and 3, and its expression of TGF-β receptors I and II were examined. We demonstrated that these tumor cells are not contact-inhibited and that they can proliferate in the absence of additional serum growth factors. In sparse cultures, all TGF-β forms inhibited the growth of HCS-2/8 cells, whereas they induced a 2-fold increase of DNA synthesis in serum-fed confluent cultures. In serum-free confluent conditions only TGF-β1 stimulated the proliferation rate, whereas TGF-β2 was without effect and TGF-β3 was rather inhibitory. This bimodal effect of TGF-β forms was associated with a greater level of TGF-β receptor I mRNA in confluent HCS-2/8 than in sparse cultures, suggesting that the growth response to TGF-β forms is dependent on the receptor profile expressed.     

The αVβ3/αVβ5 integrin inhibitor cilengitide augments tumor response to melphalan isolated limb perfusion in a sarcoma model

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)‐α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better‐tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha‐V integrins, has both anti‐angiogenic and direct anti‐tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra‐peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.     

T-Cell Receptor α and δ Gene Assembly in B-Cell Precursor Acute Lymphoblastic Leukemia

The status of the TCR-s/δ genes in B-precursor ALL and the rearrangement patterns of these gene loci are discussed in this review. Although most of these rearrangements have been characterized, some still remain to be clarified. Almost all rearrangements of the TCRs in B-precursor ALL are incomplete and may reflect early recombinational steps during the TCR differentiation processes in normal T-lineage cells. In addition, even in T-cell malignancies, it is rarely possible to obtain clonal cell populations with TCR rearrangements arrested in very early recombinational steps. Therefore, studies of these as yet uncharacterized rearrangements may lead to the discovery of additional gene segments playing important roles in the TCR recombinational processes and may provide useful information for understanding the processes of T-cell differentiation.