Immunohistochemical analysis of cyclin A in astrocytic tumours

Cyclins are important regulators of the cell cycle; there is increasing evidence that some cyclins are positively involved in carcinogenesis. Amplification and translocation of the cyclin genes and overexpression of their mRNAs and proteins have been observed in a variety of tumours. We studied cyclin A protein in astrocytic tumours by immunohistochemical analysis. Immunohistochemistry with microwave antigen retrieval was carried out on formalin fixed, paraffin embedded material from 15 glioblastomas (WHO grade IV), 10 anaplastic astrocytomas (WHO grade III), seven diffuse low grade astrocytomas (WHO grade II) and nine pilocytic astrocytomas (WHO grade I) using antibodies against cyclin A and a proliferation marker MIB1. Staining for these antibodies was seen mainly in the tumour cell nuclei; 66% of all cases showing staining for cyclin A and 95% of all cases staining for MIB1. Mean labelling indices (LI) for cyclin A were higher in glioblastoma (mean LI-6.7) and anaplastic astrocytoma (mean LI-5.9) than low grade diffuse astrocytoma (mean LI-1.7) and pilocytic astrocytoma (mean LI −0.12), although there was no clear cut off point between the various tumour types. A good correlation was seen between labelling indices of cyclin A and MIB1 (Pearson correlation coefficient r =0.59, P <0.0001). Cyclin A is variably expressed in astrocytic tumours, either reflecting increased tumour proliferation (cyclin A being an integral component of the cell cycle), an alteration of its gene, protein upregulation or regulation of apoptosis. The genetic basis of expression of cyclin A in astrocytic tumours remains to be determined.     

人脑胶质瘤中β-catenin的表达与细胞增殖相关性研究

目的:探讨人脑胶质瘤中βcatenin的表达及与细胞增殖的相关性。方法:用免疫组化法观察了47例胶质瘤标本βcatenin、cyclinD1、增殖细胞核抗原(PCNA)的表达,并分析其相关性。结果:在胶质瘤中βcatenin、cyclinD1、PCNA均随肿瘤恶性程度增高而增高(P分别为0.017、0.017、0.027),相关分析发现βcatenin、cyclinD1、PCNA的表达两两相关(P<0.001,P<0.001和P=0.005)。结论:βcatenin表达水平的异常与胶质瘤细胞周期调节紊乱、增殖指数上升有关,因此可能是控制肿瘤细胞增殖活性的候选基因之一。     

Cyclin D1 expression in normal oligodendroglia and microglia cells: Its use in the differential diagnosis of oligodendrogliomas

Cyclin D1 regulates G1–S progression. In many carcinomas it is overexpressed and it might even correlate with prognosis. However, the amplification of CCND1 contributes to the loss of cell cycle control only in a small fraction of malignant gliomas. Cyclin D1 can be immunohistochemically demonstrated by DCS‐6 mAb. In astrocytic gliomas the fraction of tumor cells with positive nuclei is almost null in well differentiated tumors and increases with the increase of proliferation rate that occurs in anaplasia. The correct evaluation of this fraction is hindered by the positive staining of normal oligodendrocytes and microglia cells. The cyclin D1‐positive staining of normal oligodendrocytes and microglia cells has been studied in a series of 20 oligodendrogliomas, five diffuse astrocytomas and five oligoastrocytomas and in 10 samples of normal cortex and white matter, using cyclin D1 DCS‐6 mAb, Feulgen reaction and CR3.43 mAb for microglia cells. As well as microglial nuclei, the nuclei of normal oligodendrocytes of the cortex and white matter, including peri‐neuronal satellites and pericapillary cells, were immunostained by DCS‐6 mAb. In infiltrative areas of oligodendrogliomas, normal, cyclin D1‐positive oligodendrocytes and cyclin D1‐negative tumor cells coexisted. In anaplastic oligodendrogliomas, cycling tumor oligodendrocytes may regain the capacity to express cyclin D1, which is thus positive in some tumor cells. The occurrence of positive oligodendrocytes in the peripheral parts of tumors can be useful in distinguishing astrocytomas from oligoastrocytomas.     

7蛋白酶体抑制剂对星形胶质细胞周期素D1和周期素依赖性激酶4表达的影响

目的：研究蛋白酶体抑制对体外培养的星形胶质细胞周期素Dl（cyclinD1）和周期素依赖性激酶4（CDK4）表达的影响。方法：SD乳鼠皮质星形胶质细胞原代培养,并纯化鉴定;予不同浓度（2和4μmol·L^-1）的蛋白酶体抑制剂（lactacystin）对第二代星形胶质细胞进行短期（12h）急性干预处理,应用免疫荧光及Westernblot检测星形胶质细胞cyclinD1和CDK4表达的水平。结果：纯化传代的皮质星形胶质细胞经胶质纤维酸性蛋白（GFAP）免疫荧光鉴定,其阳性率可达99％;lactacystin2和4μmol·L^-1可诱导星形胶质细胞cyclinDl和CDK4表达的下降,与对照组相比差异有显著统计学意义（P〈0．01）。结论：一定程度蛋白酶体活性抑制可诱导培养的星形胶质细胞cyclinD1和CDK4表达的减少,从而影响胶质细胞细胞周期,促进胶质细胞分化。提示蛋白酶体功能障碍后可能通过影响胶质细胞细胞周期来参与阿尔茨海默病的病理改变。     

Cyclin D1 expression in gliomas

Cyclin D1 (cycD1) expression was defined immunohistochemically using monoclonal antibody DCS-6 and polyclonal antiserum H-295 in 50 glioma biopsies. The number of positive nuclei was higher for H-295 than for DCS-6, with a ratio of 3:1. The labelling index (LI) was compared to the grade of histological malignancy and to Ki-67 MIB-1 LI. The LI for cycD1 increased with histological malignancy, in parallel with the increase in MIB-1 LI. In most tumours, the maximum LI for cycD1 and MIB-1 were found in the same areas. The mean MIB-1 LI: mean cycD1 LI ratio does not vary in the three grades of astrocytic tumours. However, in this study the correlation between the two LIs was not statistically significant. Staining for cycD1 antigen does not necessarily imply that the gene is overexpressed since other molecular mechanisms can also be responsible for cell cycle deregulation. In invasive areas, the cycD1 LI is frequently higher than in solid tumour, either because more tumour cells are positive or because reactive astrocytes and activated microglia express cycD1. The relative contribution of neoplastic and reactive cells remains to be defined. Received: 23 May 1997 / Revised: 21 July 1997 / Accepted: 20 August 1997     

Cyclin D1/D2/D3在大鼠脑胶质瘤组织中的表达

目的 探讨cyclin D1/D2/D3在大鼠脑胶质瘤组织中的表达.方法 将体外培养的大鼠C6胶质瘤细胞(细胞数为3×105个)借助动物立体定向仪接种于Wistar大鼠左侧尾状核区,解剖标本,行组织病理学检查及GFAP、cyclin D1/D2/D3免疫组化检测.结果 cyclinD1/D2/D3均呈阳性表达.cyclin D1/D3主要在细胞胞核表达,cyclin D2主要在胞浆表达,部分在胞核表达,不同存活期荷瘤鼠瘤组织中cyclin D1/D2/D3表达量不同.结论 cyclinD1/D2/D3过分表达参与调节大鼠脑胶质瘤增殖.荷瘤鼠生存期与cyclin D家族蛋白的表达呈负相关关系.     

Galectin-3 and galectin-3-binding site expression in human adult astrocytic tumours and related angiogenesis

Using computer-assisted microscopy, the present work aimed to quantitatively characterize the level of the histochemically detectable expression of galectin-3 and galectin-3-binding sites in sections of a series of 84 astrocytic tumours (including 22 grade II, 21 grade III and 41 grade IV specimens) and seven non-tumoural specimens used as controls. The presence of galectin-3 and reactive sites for this lectin were monitored by means of a specific polyclonal anti-galectin-3 antibody (aGal3) and biotinylated galectin-3 (Gal3), respectively. The pattern of expression of galectin-3-binding sites is compared to the pattern of expression of laminin (a potential galectin-3 ligand) revealed using a biotinylated anti-laminin antibody (aLam). Three variables quantitatively characterizing histochemical staining reactions were evaluated by means of computer-assisted microscopy for each of the 3 probes under study (aGal3, Gal3 and aLam). The labelling index (LI) is the percentage of tissue area specifically stained by a histochemical probe. The mean optical density (MOD) denotes staining intensity. The concentration heterogeneity (CH) feature expresses the concentrational spread of individual fields. The data obtained in the present study show that: (i) white matter of a non-tumoural brain expresses galectin-3 (and also galectin-3-binding sites); (ii) the level of galectin-3 expression significantly decreases in the majority of tumour astrocytes from low to high grade astrocytic tumours; while (iii) some tumour cell clones expressing high amounts of galectin-3 emerged with increasing levels of malignancy; and (iv) the level of accessible galectin-3-binding sites was apparently not heavily modified in the course of malignancy progression. In conclusion, the results obtained in the present study show that human astrocytic tumours are very heterogenous in their galectin-3 levels of expression. If high levels of galectin-3 determine the invasiveness potential of a tumour cell, then within a heterogenous tumour the presence of even a small, but actively proliferating number of tumour cell clones expressing high levels of galectin-3 can be expected to lead to tumour invasiveness.     

Prognostic indicators in a range of astrocytic tumours: an immunohistochemical study with Ki-67 and p53 antibodies.

The treatment and prognosis of patients with cerebral astrocytic tumours are currently guided by histopathological classification. This study evaluates immunohistochemistry using Ki-67, an antibody to a nuclear protein expressed in proliferating cells, and DO-7, an antibody to the product of the tumour suppressor gene p53, as prognostic indicators for these tumours. Immunohistochemistry with Ki-67 has been correlated with the behaviour of many different tumours, but its value as a prognostic indicator in astrocytic tumours is diminished by the conflicting results of previous studies. Immunohistochemistry with antibodies to the p53 protein has been used as a prognostic indicator in melanomas and some carcinomas, but the relation between prognosis and accumulation of this protein in astrocytic tumours has not been clarified. We have tested the hypothesis that survival is correlated with Ki-67 immunolabelling indices (LIs) and patterns of p53 immunolabelling in the cerebral astrocytic tumours of a large cohort of patients (n = 123) for whom clinical indices were well documented. Astrocytic tumours were divided into three histological types: fibrillary astrocytoma (n = 24), anaplastic astrocytoma (n = 31), and glioblastoma (n = 68). Histological type and patient age were independent predictors of survival. Median Ki-67 LIs differed significantly (P < 0.0001) between the types of astrocytic tumour, and tumours with a Ki-67 LI < 2% had a significantly (P < 0.0001) better prognosis. Ki-67 LI as a continuous variable carried a significant (P = 0.0043) unadjusted hazard to survival which was lost when adjusted for other variables, notably histological type. By contrast, no relation was found between survival and three categories of p53 labeling (p53-negative, p53 LI < 40%, and p53 LI > 60%). The results indicate that, whereas Ki-67 immunohistochemistry predicts survival in patients with astrocytic tumours, conventional histological appraisal remains the best guide to prognosis, and immunohistochemistry for p53 has no value in the assessment of these tumours.     

胶质瘤PTEN和细胞周期蛋白D1表达及其意义

目的　探讨胶质瘤组织中PTEN和细胞周期蛋白D1(CyclinD1)表达的意义。 方法　应用免疫组化结合计算机图像分析方法检测PTEN和CyclinD1蛋白在 80例胶质瘤的表达及其相关关系。结果　PTEN阳性率为 6 3.8% (5 1/ 80 ) ,低分级胶质瘤 (I～II)阳性率显著高于III级和IV级 (P<0 .0 1) ;胶质瘤不同病理分级间CyclinD1蛋白表达存在显著性差异 (P <0 .0 1)。胶质瘤细胞CyclinD1与PTEN蛋白表达呈显著性负相关关系 (r=- 0 .5 4 3,P <0 .0 1)。结论　PTEN蛋白表达可能在胶质瘤细胞周期调节和细胞增殖中发挥重要作用     

Characterization of perivascular cells in astrocytic tumours and peritumoral oedematous brain

Perivascular cells (PVCs) form an immunophenotypically defined population that plays an important scavenging role in the perivascular fluid drainage pathways in the rat brain: such cells may also act as antigen-presenting cells. The present study tests the hypotheses that (a) PVCs in human brain are distinct from microglia and haematogenous macrophages, and (b) PVCs within astrocytic tumours and peritumoral oedematous brain tissue react in a similar way to PVCs in the rat brain. Parafin sections of formalin-fixed tissue from 10 astrocytomas. 10 anaplastic astrocytomas, 10 glioblastoma multiforme. peritumoral oedematous brain and from normal human brain were examined immuno-cytochemically using antibodies HLA-DR P-chain for MHC class II antigen, PGMl and MAC 387 directed against macrophage components, MT1 for T lymphocytes and GFAP for astrocytes. No PVCs, microglia or macrophages were labelled by these techniques in paraffin sections of normal brain. Microglia. Macrophages recently derived from haematogenous monocytes and PVCs were labelled by immunocytochemistry in all tumours but were more numerous in glioblastomas than in astrocytomas or anaplastic astrocytomas. Perivascular cells were distinguished by their perivascular position, their expression of MHC class II antigen and were labelled by PGM1 antibody but not by MAC 387 antibody. Microglia and monocyte/macrophages, remote from blood vessels, on the other hand, were strongly labelled by MAC 387, moderately by PGMl and showed weak expression of MHC class II antigen. A similar pattern of staining was seen in peritumoral oedematous tissue. These findings suggest that PVCs form a defined population of resident cells in the human brain and that they are distinct from microglia, monocytes and macrophages. Furthermore. upregulation of MHC class II and PGM1 expression on PVCs in tumours and oedematous brain, suggest that they play a similar scavenging role in the human brain to that seen in the rat brain.     

胶质细胞瘤复发前后Cyclin D1和P16的表达

目的　探讨CyclinD1,P16在胶质瘤复发前后表达改变及其意义。方法　采用免疫组织化学LsABC法对 4 5例复发胶质瘤瘤组织、瘤旁脑组织和 10例正常脑组织CyclinD1,P16蛋白表达进行检测 ,统计分析CyclinD1,P16表达水平与胶质瘤分级、肿瘤复发的关系。结果　正常脑组织 ,瘤旁脑组织和胶质瘤组织CyclinD1表达依次升高 ,而P16的表达依次下降 ;肿瘤复发CyclinD1表达增强 ,P16的表达减弱。结论　CyclinD1与P16的表达与胶质瘤恶性进程和复发密切相关。     

Differential polymerase chain reaction and immunohistological studies on amplification and expression of cyclin D1 gene in human glioma tissues

Using differential polymerase chain reaction we studied the amplification of the cyclin D1 gene in formalin-fixed, paraffin-embedded surgical specimens of 70 gliomas. We also examined expression of cyclin D1 in these gliomas, and the Ki-67 labeling indices in 24 glioblastomas by immunohistochemistry. Among 70 gliomas, amplification of cyclin D1 gene was found in only one glioblastoma. The expression of cyclin D1 was immunohistochemically detected in only one glioblastoma with cyclin D1 gene amplification. The highest labeling index of Ki-67, 48.0%, was noted in one of cyclin D1 positive glioblastoma (the average labeling index of 30.2% in cyclin D1 negative glioblastomas). These results suggest that amplification of cyclin D1 gene could play a role in the development of glioblastoma but it is unlikely to be the main gene alteration in malignant progression of gliomas in the majority of cases.     

Immunohistochemical expression of cyclin D1 is higher in supratentorial ependymomas and predicts relapses in gross total resection cases

Ependymomas are tumors of the CNS. Although cyclin D1 overexpression has been related to several cancers, its prognostic value in ependymomas has not yet been fully established. We evaluated cyclin D1 expression by an immunohistochemistry analysis of 149 samples of ependymomas, including some relapses, corresponding to 121 patients. Eighty‐one patients were adults, 60 were intracranial cases and 92 tumors were grade II. Gross total resection (GTR) was achieved in 62% of cases, and relapse was confirmed in 41.4% of cases. Cyclin D1 protein expression was analyzed by immunohistochemistry and scored with a labeling index (LI) calculated as the percentage of positively stained cells by intensity. We also analyzed expression of CCND1 and NOTCH1 in 33 samples of ependymoma by quantitative real‐time PCR. A correlation between cyclin D1 LI score and anaplastic cases (P < 0.001), supratentorial location (P < 0.001) and age (P = 0.001) were observed. A stratified analysis demonstrated that cyclin D1 protein expression was strong in tumors with a supratentorial location, independent of the histological grade or age. Relapse was more frequent in cases with a higher cyclin D1 LI score (P = 0.046), and correlation with progression‐free survival was observed in cases with GTR (P = 0.002). Only spinal canal tumor location and GTR were suggestive markers of PFS in multivarite analyses. Higher expression levels were observed in anaplastic cases for CCND1 (P = 0.002), in supratentorial cases for CCND1 (P = 0.008) and NOTCH1 (P = 0.011). There were correlations between the cyclin D1 mRNA and protein expression levels (P < 0.0001) and between CCND1 and NOTCH1 expression levels (P = 0.003). Higher cyclin D1 LI was predominant in supratentorial location and predict relapse in GTR cases. Cyclin D1 could be used as an immunohistochemical marker to guide follow‐up and treatment in these cases.     

A study of glutamine synthetase in normal human brain and intracranial tumours

Glutamine synthetase (GS) activity was measured in selected areas of three normal brains and in 262 biopsies from patients with suspected intracranial tumours. In general, levels were higher in grey matter than in white matter and the highest activities of all were found in the hypothalamus which is consistent with its high glutamatergic activity. In the biopsy material, GS activity was greatest in gliotic brain, in keeping with the predominantly astrocytic localization of the enzyme. High levels were also found in astrocytomas and oligodendrogliomas but there was considerable variation between tumours, suggesting a random loss of GS expression during neoplastic transformation or heterogeneity in their cellular origin. The immunocytochemical demonstration of GS in neoplastic oligodendrocytes and in meningioma cells argues against absolute cell-type specificity for this enzyme.     

Cyclin D1 in excitatory neurons of the adult brain enhances kainate-induced neurotoxicity

G1-phase cyclin D1 (cD1) expression has been documented in post-mitotic neurons undergoing apoptosis, leading others to propose that attempted cell cycle re-entry may induce cell death. Here, cD1 immunoreactivity was found in a subpopulation of healthy excitatory neurons throughout the brain. Most striking was the selective cD1 expression in hippocampal pyramidal neurons, an especially vulnerable cell group. Seizure threshold, cD1 induction and CA1 neuron death were examined following application of kainate (KA) or pentylenetetrazole (PTZ) in cD1 heterozygous (+/−) and wildtype mice to determine whether baseline cD1 correlates with pathology. cD1+/− mice displayed resistance to KA, but not PTZ-induced seizures and had reduced or equivalent cytotoxicity respectively, compared with wildtype. KA administration, but not PTZ, induced cD1 expression. These findings suggest that basal cD1 expression may render hippocampal circuits more susceptible to particular epileptogenic agents and excitotoxic cell death, though cD1 is not a direct precipitant in apoptosis.     

Short communication CNS tumours: oligoclonal immunoglobulin D in cerebrospinal fluid and serum

Oligoclonal immunoglobulin D bands were detected by isoelectric focusing in 7 out of 25 unconcentrated cerebrospinal fluid (CSF) samples obtained from patients with tumours of the central nervous system (CNS). The tumours were confirmed by clinical and histological findings. Two patients with CNS malignancy had intrathecal synthesis of oligoclonal bands both IgD and IgG. Four patients with a variety of CNS tumours had a systemic IgD immune response but no oligoclonal IgG bands. One patient with the most malignant tumour histology had a systemic IgD response as well as local synthesis of IgG. The study reveals several new aspects regarding CNS tumours: they are immunologically active and are capable of invoking oligoclonal immunoglobulin production both within the CNS and systemically. Multiple immunoglobulin activation can be found in malignant CNS tumours, and systemic IgD production may occur independently from IgG synthesis and may represent an immune response to a neoantigen produced in the CNS compartment.     

Epidermal growth factor receptor expression and activation in neuroendocrine tumours

Epidermal growth factor receptor (EGFR) is expressed in many cancers and is associated with poor prognosis. EGFR activation pathways have been well characterised using tumour cell lines and are known to involve EGFR activation through autophosphorylation. Phosphorylation of downstream signalling molecules, such as ERK1/2 (extra-cellular regulated kinase 1 and 2) and PKB/Akt (protein kinase B), leads to enhanced tumour cell survival and proliferation. Although EGFR expression has been determined in neuroendocrine tumour tissue, its activation and subsequent effects on the downstream signalling molecules, ERK1/2 and Akt, have not been studied. We therefore planned to determine the role of EGFR in neuroendocrine tumours (NETs) by determining its pattern of expression and activation, and the subsequent activation of downstream signalling molecules ERK1/2 and Akt. Paraffin-embedded tumour tissue was available from 98 patients with NETs (39 foregut, 42 midgut, four hindgut, five paragangliomas, and four of unknown origin). Immunohistochemical evaluation was performed for the expression of EGFR, p-EGFR, p-Akt, and p-ERK1/2. Ninety-six percent of tumour samples were positive for EGFR expression; 63% were positive for activated EGFR; 76% were positive for activated Akt; and 96% were positive for activated ERK1/2. Importantly, the histological score for the activation of Akt and ERK1/2 correlated with the histological score for activated EGFR. These data provide a rationale for considering EGFR inhibitors in the treatment of NETs. Additionally, direct inhibition of Akt and ERK1/2 may provide further therapeutic options in the treatment of NETs in the future.     

Serum and forskolin cooperate to promote G1 progression in Schwann cells by differentially regulating cyclin D1, cyclin E1, and p27Kip expression

Proliferation of Schwann cells in vitro, unlike most mammalian cells, is not induced by serum alone but additionally requires cAMP elevation and mitogenic stimulation. How these agents cooperate to promote progression through the G1 phase of the cell cycle is unclear. We studied the integrative effects of these compounds on receptor-mediated signaling pathways and regulators of G1 progression. We show that serum alone induces strong cyclical expression of cyclin D1 and E1, 6 and 12 h after addition, respectively. Serum also promotes strong but transient erbB2, ERK, and Akt phosphorylation, but Schwann cells remain arrested in G1 due to high levels of the inhibitor, p27(Kip). Forskolin with serum promotes G1 progression in 22% of Schwann cells between 18 and 24 h by inducing a steady decline in p27(Kip) levels that reaches a nadir at 12 h coinciding with peak cyclin E1 expression. Forskolin also delays neuregulin-induced loss of erbB2 receptors allowing strong acute activation of PI3K, sustained erbB2 phosphorylation and G1 progression in 31% of Schwann cells. We find that the ability of forskolin to decrease p27(Kip) is associated with its ability to decrease Krox-20 expression that is induced by serum and further increased by neuregulin. Our results explain why serum is required but insufficient to stimulate proliferation and identify two routes by which forskolin promotes proliferation in the presence of serum and neuregulin. These findings provide insights into how G1 progression and, cell cycle arrest leading to myelination are regulated in Schwann cells.     

Neuronal integrity and astrocytic reaction in cold injury: an immunohistochemical investigation

The relationship between extravasation of serum albumin and damage to the neuronal elements as well as the astrocytic reaction was investigated following cold injury, using immunohistochemistry for albumin, microtubule-associated protein I and II (MAPs) and glial fibrillary acidic protein (GFAP). After 30 min, spreading of albumin to the neuropil and uptake into nerve cell bodies and dendrites were clearly observed in the area surrounding the cold lesion. Extravasation of albumin was maximal at 24 h and extended to the ipsilateral hippocampus and thalamus as well as to the paramedian part of the contralateral cerebral hemisphere. Uptake of albumin was seen in neurons with and without loss or reduction of the reaction for MAPs, but the former was confined to the area surrounding the cold lesion. When extravasated albumin receded from the neuropil, the positive reaction for albumin also disappeared from the neuronal elements and those neurons recovered immunoreactivity for MAPs. Astrocytes immunopositive for albumin were observed at 24 h in the white matter, and reactive astrocytes became notable even in the gray matter surrounding the cold lesion. Although reactive astrocytes persisted even after resolution of cerebral edema, immunopositivity for albumin disappeared from astrocytes soon after the disappearance of the reaction from the neuropil. As to the mechanism, rapid endo-and exocytosis may take place in response to the amount of edema fluid in the surrounding extracellular space, where albumin may be eliminated through the transvascular route and/or via the cerebrospinal fluid space. Received: 26 August 1996 / Revised, accepted: 15 January 1997     

Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures

Astrocyte activity may be modulated by steroid hormones and GFs. This study investigates the interaction between glucocorticoids or estrogens and GFs on the expression of heme oxygenase‐1 (HO‐1) and cyclin D1 in astrocyte cultures at 14 days treated for 48 or 60 hr with dexamethasone (DEX) or 48 hr with 17β‐estradiol (E₂) alone or with GFs added only in the last 12 or 24 hr. Twelve‐ or twenty‐four‐hour epidermal growth factor (EGF) treatment significantly enhanced HO‐1 expression in astrocyte cultures pretreated for 48 hr with DEX. A highly significant increase in HO‐1 expression was obtained after the last‐12‐hr EGF treatment in 48‐hr E₂‐pretreated astrocyte cultures; this enhancement was particularly significant in 48‐hr E₂‐pretreated cultures as well as in the last‐12‐hr insulin‐treated ones pretreated for 48 hr with E_2. Sixty‐hour DEX‐alone pretreatment as well as the last‐12‐hr EGF treatment in 60‐hr DEX‐pretreated astrocyte cultures showed a significant increase of cyclin D1 expression. A significant decrease of cyclin D1 expression in the last‐12‐hr insulin‐like growth factor‐I (IGF‐1)‐treated cultures pretreated for 60 hr with DEX was observed. A highly significant enhancement in cyclin D1 expression in 14 days in vitro astrocyte cultures pretreated with E₂ alone for 48 hr and treated in the last 12 hr with IGF‐1 in 48‐hr E₂‐pretreated cultures was found. Finally, the data highlight an interactive dialogue between the growth factors and glucocorticoids or estrogens during the maturation of astroglial cells in culture that may control the HO‐1 and cyclin D1 expression as well as proliferating astroglial cells during the cell cycle. © 2014 Wiley Periodicals, Inc.