Inhibition of granulocytic differentiation of acute promyelocytic leukemia cells in primary culture by transforming growth factor-β

We investigated the effect of transforming growth factor-β₁ (TGFβ) on the proliferation and differentiation of cultured acute promyelocytic leukemia (APL) cells with the chromosomal t(15;17) translocation obtained from four patients to determine the role of TGFβ on growth and differentiation of APL cells.

DNA synthesis, determined by ³H-thymidine uptake, was inhibited in the presence and absence of granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner by TGFβ in APL cells obtained from three of the four cases. TGFβ and G-CSF did not significantly affect the differentiation of APL cells, but all-trans retinoic acid (RA) induced morphological and functional differentiation in all APL cells tested. G-CSF markedly enhanced RA-induced granulocytic differentiation in APL cells obtained from all four cases. In cells in which TGFβ inhibited DNA synthesis, it also inhibited RA-induced granulocytic differentiation of APL cells and, to a greater degree, granulocytic differentiation induced by RA plus G-CSF.

These results suggest that TGFβ is a negative regulator of the proliferation and differentiation of APL cells. The significance of TGFβ as an endogenous regulator in differentiation therapy with RA of APL patients is discussed.     

Altered expression of transforming growth factor-β1 mrna and protein in mouse skin carcinogenesis

Transforming growth factor (TGF)-β1, whose gene is located on mouse chromosome 7, has been proposed to be involved in skin carcinogenesis. In the study presented here, we demonstrated that single topical treatments with different types of tumor promoters, i.e., the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA, 2 μg); the non–protein kinase C activators anthralin (22.6 μg), benzoyl peroxide (20 mg), and cumene hydroperoxide (1.2 mg); the first-stage tumor promoters 4-O-methyl-TPA (500 μg) and A23187 (166 μg); and the second-stage tumor promoter mezerein (2 μg) produced transient induction of TGF-β1 mRNA in SSIN (inbred SENCAR) mouse skin. The time of maximum induction varied from 3 to 12 h; the relative extent of induction was ranked as cumene hydroperoxide > benzoyl peroxide > anthralin > TPA > 4-O-methyl-TPA > mezerein > A23187. These findings suggested that TGF-β1 mRNA induction is a common response of skin to several types of complete and stage-specific promoters; however, the extent of induction did not correlate with the reported hyperplastic activity of single applications of these promoters. We also demonstrated that TGF-β1 mRNA expression in papillomas of SENCAR mice generally correlated with expression levels of cyclin D1, another gene on chromosome 7, and with stage of tumor progression. TGF-β1 mRNA expression was constitutively elevated in most squamous cell carcinomas from either initiation-promotion or complete carcinogenesis protocols. Cell lines established from carcinomas also overexpressed TGF-β1 mRNA. Immunohistochemical staining of tissue sections of normal and TPA-treated skin revealed the presence of extracellular TGF-β1 protein in the dermis and intracellular TGF-β1 protein in the epidermis, especially in the suprabasal layers. The staining patterns of papillomas varied, with 62 ± 13% of the tissue showing strong intracellular staining but only 25 ± 8% of the connective tissue staining for extracellular TGF-β1. Variable staining patterns were also found in carcinomas; some areas stained heavily for both the intracellular and extracellular forms of TGF-β1. Overall, 28 ± 6% of the tissue of the 12 analyzed carcinomas stained for the intracellular form and 18 ± 5% for the extracellular from of TGF-β1. © 1994 Wiley-Liss, Inc.     

Phase I study of hypofractionated dose-escalated thoracic radiotherapy for limited-stage small-cell lung cancer

PURPOSE: To determine the maximal tolerated dose of hypofractionated thoracic radiotherapy with concurrent chemotherapy for limited-stage small-cell lung cancer patients. METHODS AND MATERIALS: Three radiotherapy regimens were used. Radiotherapy was given in two phases: patients initially received 20 Gy in 10 fractions to gross tumor plus uninvolved mediastinal nodes, followed by a boost to gross disease of 30, 38, or 42 Gy in 15 fractions. Radiotherapy was planned with conformal techniques. All patients received four cycles of cisplatin (25 mg/m2) and etoposide (100 mg/m2) chemotherapy. Radiotherapy commenced with Day 1 of Cycle 2 of chemotherapy. All complete/near-complete responders were offered prophylactic cranial irradiation. The maximal tolerated dose of radiotherapy was based on the dose that caused unacceptably high rates of radiotherapy-related toxicity. RESULTS: Thirteen patients were accrued. All patients who commenced radiotherapy received all prescribed chemo- and radiotherapy. There were no treatment-related deaths. There was one Grade 3 acute nonhematologic toxicity in the 50-Gy group. Of the 6 patients given 58 Gy, 3 experienced acute Grade 3 esophagitis. With a median follow-up of 7 months, median overall survival was 9.5 months. CONCLUSIONS: The maximal tolerated dose of thoracic radiotherapy with concurrent chemotherapy on this trial was 50 Gy in 25 daily fractions.     

Contemporary brachytherapy approaches in non–small-cell lung cancer

Brachytherapy has the ability to deliver a higher tumor dose compared to external beam irradiation, while sparing normal tissue outside the tumor; it is the most effective means of delivering conformal radiation and can be tailored to clinical circumstances, either at open surgery or in an ambulatory setting, which is currently the preferred method. Intraoperative lung and/or endobronchial brachytherapy in the management of non–small-cell lung cancer offers a good curative potential in patients with accessible localized tumors, well defined and small to moderate in size, that have not metastasized to the lymph nodes and are technically or medically inoperable. Effective palliation can be frequently obtained by endobronchial brachytherapy on an outpatient procedure basis. Brachytherapy administered simultaneously with chemotherapy is better tolerated than a course of external beam irradiation and chemotherapy. J. Surg. Oncol. 1998;69:258–264. © 1998 Wiley-Liss, Inc.     

Dose- and time-dependent expression of transforming growth factor-β1 mRNA and protein in mouse epidermis and papillomas after repeated topical application of benzo[a]pyrene

Topical weekly application of 64 μg of benzo[a]pyrene (BAP) for 4 wk induced transforming growth factor (TGF)-β1 mRNA in the epidermis of Swiss (ICR) mice, with a maximum at 6–12 h after the last treatment. The increase in TGF-β1 mRNA concentration was accompanied by an increase in immunohistochemically detectable intracellularly localized TGF-β1 protein in the suprabasal epidermis and by the appearance of extracellularly localized TGF-β1 in the basal layers. A dose rate of 16 μg/wk for 4 wk was unable to induce the same response. In contrast, after 20 weekly topical applications of 16 or 64 μg of BAP, an increase in TGF-β1 mRNA concentration and the appearance of extracellularly localized protein in the epidermis were observed. These changes in TGF-β1 expression were paralleled by changes in epidermal morphology. A similar group of animals treated with 4 μg of BAP/wk for 20 wk did not respond differently from untreated controls. Papillomas resulting from treatment with 16 or 64 μg of BAP/wk for 28 wk stained for intracellularly localized TGF-β1 predominantly in the differentiating and nondividing layers. Papillomas stained for extracellularly localized TGF-β1 solely in the less differentiated and dividing cells. These results suggest that tumorigenesis by BAP involves the induction of cumulative changes in epidermal TGF-β1 mRNA and protein concentrations as well as alterations in skin morphology associated with a tumor-promotion process.     

Transforming growth factor-β1 expression in Syrian hamster cheek pouch carcinogenesis

The expression pattern of transforming growth factor-β1 (TGF-β1) during the stages of complete carcinogenesis in the hamster cheek pouch model was studied. The right cheek pouches of 18 male hamsters were treated with 0.5% 7,12-dimethylbenz[a]anthracene (DMBA) for 16 wk. TGF-β1 was detected immunohistochemically in the resulting samples with two different polyclonal monospecific antibodies that recognize intracellular and extracellular forms of TGF-β1. In the normal cheek pouch, extracellular protein stained the corium strongly, but the reaction was not evenly distributed. As treatment progressed, the reaction increased in both area and intensity; the peak was reached at 8 wk. Intracellular TGF-β1 expression followed a similar pattern, with a peak at 4 wk of treatment. The results of northern blot analysis were concordant with the immunohistochemical results. Overexpression of TGF-β1 was also observed in the malignant tumors, but only the extracellular form of the protein was present; intracellular TGF-β1 was not detected in these tumors. The expression of TGF-β1 in this carcinogenesis model seems to have two formal stages, the first being an overexpression step as a reaction to the uncontrolled growth and the second being one in which tumors have no internal expression of TGF-β1 but in which external protein accumulates in the surrounding stroma. A possible explanation of this paradox may be that TGF-β1 has functions other than its growth-repressing activity. © 1994 Wiley-Liss, Inc.     

Final results of a Phase I/II dose escalation trial in non-small-cell lung cancer using three-dimensional conformal radiotherapy

PURPOSE: The aim of this study was to determine the maximum tolerated dose (MTD) delivered within 6 weeks in patients with non-small-cell lung cancer (NSCLC). The impact of tumor volume and delivered dose on failure-free interval (FFI) and overall survival (OS) were also studied. METHODS AND MATERIALS: A Phase I/II trial was performed including inoperable NSCLC patients. According to the relative mean lung dose (rMLD), five risk groups with different starting doses were defined: Group 1, rMLD 0.0 to 0.12; Group 2, rMLD 0.12 to 0.18; Group 3, rMLD 0.18 to 0.24; Group 4, rMLD 0.24 to 0.31; and Group 5, rMLD 0.31 to 0.40. Patients underwent irradiation with 2.25 Gy per fraction and a fixed overall treatment time of 6 weeks. The dose was escalated with 6.75 Gy after 6 months follow-up without dose-limiting toxicity. If more than 30 fractions were prescribed, twice-daily irradiation was performed with at least a 6-h interval. RESULTS: A total of 88 patients were included. Tumor Stage I or II was found in 53%, IIIA in 31%, and IIIB in 17%. The MTD was not achieved in risk Group 1 (reached dose, 94.5 Gy). For risk Groups 2 and 3 the MTD was 81 Gy. The 74.3-Gy dose was determined to be safe for Group 4 and the 60.8-Gy dose for Group 5. In 2 patients (5%) an isolated nodal relapse occurred. Based on multivariable analysis, higher doses significantly increased the FFI (p = 0.02) for the total group. The OS was increased in the lower risk groups (p = 0.05) but not in the higher risk groups (p = 0.4). CONCLUSION: Dose escalation is safe up to 94.5 Gy in 42 fractions in 6 weeks in patients with an MLD 13.6 Gy or less. Higher doses are associated with a better FFI and OS for smaller tumor volumes. Involved-field irradiation results in a low percentage of isolated nodal relapses.     

Significance of transforming growth factor β1 as a new tumor marker for colorectal cancer

Transforming growth factor β1 (TGF‐β1) is thought to be involved in cancer growth and progression. TGF‐β1 changes to its active form after being secreted in its latent form. Our aim was to clarify the significance of plasma concentrations of active and total TGF‐β1 of patients with colorectal cancer. Plasma concentrations of active and total TGF‐β1 in 45 patients with colorectal cancer and 23 healthy volunteers were measured using ELISA and the activation rate (ratio of active to total TGF‐β1) was determined. Plasma concentrations of active TGF‐β1 (21.9 ± 12.8 pg/ml) were significantly higher in patients with colorectal cancer than in healthy volunteers (9.9 ± 5.9 pg/ml; p < 0.001, Welch's t‐test). Concentration of total TGF‐β1 was also significantly higher for patients with colorectal cancer (18.0 ± 13.0 ng/ml vs. 11.1 ± 6.4 ng/ml; p < 0.01, Welch's t‐test). However, there was no significant difference in the TGF‐β1 activation rate between the 2 groups. There was a correlation between Dukes' stage and plasma concentration of active or total TGF‐β1 (p < 0.01, Spearman's rank correlation test) and on day 7 the active TGF‐β1 levels for patients recovering from curative resection were similar to those of the control group of healthy volunteers. These results suggest that active TGF‐β1 might be used as a tumor marker for colorectal cancer. © 2001 Wiley‐Liss, Inc.     

Liver regeneration and hepatocarcinogenesis in transforming growth factor-α-targeted mice

Transforming growth factor-α (TGFα), a member of the epidermal growth factor receptor ligand family, has been implicated in the regeneration and transformation of liver. Our recent development of mice that are homozygous for a disrupted TGFα gene allowed us to assess the requirement for this growth factor in these complex processes. We report here that although a 70% hepatectomy produced a significant increase in hepatic TGFα protein levels in wild-type mice, liver regeneration nevertheless proceeded normally in the absence of the growth factor. The hepatocyte labeling indices determined for homozygous targeted and wild-type mice at 36 and 48 h after hepatectomy were comparable, and the total liver DNA to body weight ratios 8 d after hepatectomy were essentially identical for the two genotypes. These results indicate that TGFα is not necessary for liver regeneration. To test its requirement in liver carcinogenesis, young mice were administered single doses of diethylnitrosamine (DEN) with or without subsequent chronic treatment with the promoting agent phenobarbital (PB). Both wild-type and homozygous mutant male mice treated with DEN or DEN plus PB developed multiple preneoplastic foci or tumors by 9 mo of age with relatively high incidence. However, while five of 88 tumors in wild-type mice attained a diameter greater than 5 mm and were classified as hepatocellular carcinomas, none of 132 tumors in livers of targeted mice reached this size. Furthermore, three of these large wild-type tumors expressed significantly elevated levels of TGFα protein compared with normal liver. These results indicate that TGFα is not required for early events in chemically induced hepatocarcinogenesis but suggest that it could be important in the progression from small preneoplastic foci to large tumors. © 1996 Wiley-Liss, Inc.     

三维适形放射治疗高龄肺癌30例临床分析

目的 探讨三维适形放射治疗(three-dimensional conformal radiotherapy，3D-CRT)治疗Ⅰ～Ⅲ期高龄非小细胞肺癌的疗效及副作用。方法 对30例行3D-CRT治疗的Ⅰ～Ⅲ期高龄非小细胞肺癌进行回顾性分析。30例患者共设46个靶区。靶区剂量归一到等中心处，分割剂量3～5Gy，总量42～66Gy，3～5次／周，周剂量≤15Gy。结果 总1、2、3年生存率及中位生存期分别为65．8％、41、2％、20、6％、23个月。肿瘤1、2、3年局部控制率分别59．8％、31．1％、28．3％。放射性肺炎发生率为16．7％(5／30)，其中≥3级放射性肺炎发生率为6．7％(2／30)，1例因放射性肺炎而死亡。放射性肺纤维化发生率为10、0％(3／30)，均为1～2级。放射性食管炎的发生率分别为43．3％(13／30)．均为1～2级。结论 利用3D-CRT治疗提高肿瘤剂量，从而提高高龄肺癌的生存率，并发症低，是治疗高龄肺癌的较好手段。     

Platelet PDGF and TGF-β Levels in Myeloproliferative Disorders

Myeloproliferative disorders mainly including essential thrombocythemia, polycythemia vera, chronic myeloid leukemia and myelofibrosis with myeloid metaplasia are clonal myeloproliferative diseases in which myelofibrosis is commonly observed. The pathogenesis of myelofibrosis still remains unclear. However it was proposed that an inappropriate release of PDGF from either megakaryocytes in bone marrow or platelets in circulation might promote medullary fibrosis. Recently the role of another peptide growth factor, namely TGF-β, in the fibrotic process was emphasized. This review will focus on the different studies aimed at the evaluation of intraplatelet PDGF and TGF-β content in patients with the various MPD, and outline the salient results lending support to the potential role of PDGF and TGF-β in the promotion of myelofibrosis.     

Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor β‐induced extracellular matrix production in lung cancer cells

Activation of transforming growth factor β (TGF‐β) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial‐mesenchymal transition (EMT). Hypoxia‐inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF‐1α expression by protein phosphatase activity on dissociation of the E‐cadherin/β‐catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF‐β. By using lung cancer cells treated with HIF‐1α stabilizers or carrying doxycycline‐dependent HIF‐1α deletion or point mutants, we investigated the role of stabilized HIF‐1α expression on TGF‐β‐induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF‐β and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF‐1α expression was inhibited. Stabilized HIF‐1α protein expression inhibited the TGF‐β‐stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF‐1α‐induced repression of the TGF‐β‐stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF‐1α expression on inhibition of TGF‐β‐induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.     

Regulatory role of the transforming growth factor-β signaling pathway in the drug resistance of gastrointestinal cancers

Gastrointestinal (GI) cancer, including esophageal, gastric, and colorectal cancer, is one of the most prevalent types of malignant carcinoma and the leading cause of cancer-related deaths. Despite significant advances in therapeutic strategies for GI cancers in recent decades, drug resistance with various mechanisms remains the prevailing cause of therapy failure in GI cancers. Accumulating evidence has demonstrated that the transforming growth factor (TGF)-β signaling pathway has crucial, complex roles in many cellular functions related to drug resistance. This review summarizes current knowledge regarding the role of the TGF-β signaling pathway in the resistance of GI cancers to conventional chemotherapy, targeted therapy, immunotherapy, and traditional medicine. Various processes, including epithelial-mesenchymal transition, cancer stem cell development, tumor microenvironment alteration, and microRNA biogenesis, are proposed as the main mechanisms of TGF-β-mediated drug resistance in GI cancers. Several studies have already indicated the benefit of combining antitumor drugs with agents that suppress the TGF-β signaling pathway, but this approach needs to be verified in additional clinical studies. Moreover, the identification of potential biological markers that can be used to predict the response to TGF-β signaling pathway inhibitors during anticancer treatments will have important clinical implications in the future.     

Dose-volume histogram analysis as predictor of radiation pneumonitis in primary lung cancer patients treated with radiotherapy

PURPOSE: To determine the relationship between various parameters derived from lung dose-volume histogram analysis and the risk of symptomatic radiation pneumonitis (RP) in patients undergoing radical radiotherapy for primary lung cancer. METHODS AND MATERIALS: The records of 156 patients with lung cancer who had been treated with radical radiotherapy (>/=45 Gy) and for whom dose-volume histogram data were available were reviewed. The incidence of symptomatic RP was correlated with a variety of parameters derived from the dose-volume histogram data, including the volume of lung receiving 10 Gy (V(10)) through 50 Gy (V(50)) and the mean lung dose (MLD). RESULTS: The rate of RP at 6 months was 15% (95% confidence interval 9-22%). On univariate analysis, only V(30) (p = 0.036) and MLD (p = 0.043) were statistically significantly related to RP. V(30) correlated highly positively with MLD (r = 0.96, p < 0.001). CONCLUSION: V(30) and MLD can be used to predict the risk of RP in lung cancer patients undergoing radical radiotherapy.     

Vasohibin‐2 is required for epithelial–mesenchymal transition of ovarian cancer cells by modulating transforming growth factor‐β signaling

Vasohibin‐2 (VASH2) is a homolog of VASH1, an endothelium‐derived angiogenesis inhibitor. Vasohibin‐2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial–mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor‐β (TGF‐β) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF‐β type I receptor, namely activin receptor‐like kinase 5. Transforming growth factor‐β1‐induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF‐β1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF‐β1‐induced reduced expression of epithelial markers including E‐cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF‐β1‐induced EMT. In accordance with these results, the effects of TGF‐β1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF‐β signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.     

小细胞肺癌的三维适形放射治疗

目的 分析三维适形放射治疗（3DCRT）在小细胞肺癌（SCLC）治疗中的可行性、效果和放射损伤情况。方法 19例SCLC患者中,局限期18例,广泛期1例。18例采用放化疗综合治疗,1例采用单独放疗。放疗单次剂量2Gy,每周5次,中位总剂量54Gy。化疗多采用卡铂或顺铂＋VP-16为主的方案,4～6个周期。中位随访24个月。结果 （1）全组患者CR率为31．6％（6／19）,PR率为47．4％（9／19）,SD率为21．1％（4／19）,有效率为79．0％。1年总生存率（OS）为71、7％,2年OS为35．8％,中位生存时间为19个月。1,2年无局部进展生存率均为94．7％。（2）2级急性放射性肺损伤为5．3％（1／19）,2级晚期放射性肺损伤为5．3％（1／19）,2级急性放射性食管损伤10．5％（2／19）,2级血液学毒性为10．5％（2／19）。结论 3DCRT用于SCLC治疗是可行的,患者能够获得较好的近期疗效和2年生存率,SCLC的三维适形放射治疗值得进一步研究。