Stress-response (heat-shock) protein 90 expression in tumors of the central nervous system: an immunohistochemical study

This retrospective study deals with the expression of stress-response (heat-shock) protein 90 (srp 90) in a series of 148 human brain tumors. Immunohistochemical procedures were employed; cells of the human breast cancer line MCF 7 exposed to hyperosmolar stress served as positive controls. Deposits of reaction products were found in the cytoplasm and they displayed a granular pattern. srp 90 was detected in 14/31 meningiomas and 5/10 breast cancer metastases to the brain. The protein was also present in 6/13 glioblastomas and 7/18 astrocytomas. In addition, a positive reaction was found in 2/10 medulloblastomas, 2/14 primitive neuroectodermal tumors, 1/11 pituitary tumor, 2/21 schwannomas and 2/11 lung tumor metastases; however, oligodendrogliomas and primary malignant lymphomas were not stained. The srp 90 was detected in Western blots of meiningioma tissue homogenates. No significant immunohistochemical reaction was seen with sections of normal human cerebra, brain stem, cerebella, pituitary glands and spinal cords. These results document the expression of srp 90 by a variety of primary and metastatic intracranial tumors.     

Stress-response (heat-shock) protein 72 expression in tumors of the central nervous system: an immunohistochemical investigation

Summary This report deals with the expression of stress-response (heat-shock) protein 72 (srp 72) in a series of 95 primary human brain tumors and 21 carcinoma metastases to the central nervous system (CNS). Immunohistochemical procedures were employed; cells of the human cervical cancer line HeLa S3 were used as positive controls. The protein was detected in 14/22 meningiomas and in 6/13 glioblastomas. Tumor cells expressing srp 72 were also found in 4/17 astrocytomas, 2/9 pituitary tumors, 2/14 primitive neuroectodermal tumors and 1/10 medulloblastomas. Whereas the majority (8/10) of the breast carcinoma metastases had tumor cells that expressed srp 72, only 2/11 lung tumor metastases were positively stained. These results document srp 72 expression by a variety of primary and metastatic tumors of the CNS.Supported in part by NIH grant CA47512 (to FH)     

Monoclonal antibody to human midkine reveals increased midkine expression in human brain tumors.

We produced a rat IgG2a monoclonal antibody against the carboxyl terminal region of human midkine (MK), a novel growth factor. This monoclonal antibody was used in immunohistochemical studies to compare the expression of MK, proliferating cell nuclear antigen (PCNA) and p53 protein in 133 primary brain tumors and 21 carcinoma metastases to the central nervous system. Approximately half of the glioblastomas multiforme (GBMs) (19/32), medulloblastomas (8/14), primitive neuroectodermal tumors (PNETs) (5/11), breast carcinoma metastases (Br-Mts) (6/10) and lung carcinoma metastases (L-Mts) (5/11) as well as some astrocytomas (2/14) had tumor cells that expressed MK; however, oligodendrogliomas, ependymomas, schwannomas, meningiomas, and pituitary adenomas did not express MK. The values of the PCNA-labeling index were statistically higher in GBMs, medulloblastomas, PNETs, Br-Mts, and L-Mts that expressed MK than in those that did not (Wilcoxon rank-sum test, p < 0.05). There was no correlation between MK and p53 protein in all tumor types. Normal and non-neoplastic brain tissues were negative for MK, PCNA, and p53 protein. We conclude that primary and metastatic tumors of the brain express MK and that the MK expression in brain tumors may depend, in part, on the proliferating potential.     

肿瘤转移抑制基因KiSS-1在乳腺癌脑转移患者的表达及临床意义

目的 转移抑制基因KiSS-l在多种肿瘤的浸润转移中起着重要的作用。但该基因与乳腺癌脑转移的关系仍很不清楚。本研究检测了乳腺癌原发灶和脑转移灶中KiSS-l基因的表达并探讨其临床意义。方法 选择2002年6月～2004年6月行乳腺癌脑转移病灶切除的患者12例，应用实时荧光定量PCR检测乳腺癌原发灶和转移灶中KiSS-l基因mRNA的表达，应用Westernblot检测KiSS-l蛋白的表达，并进一步应用免疫组化进行验证。结果 实时荧光定量PCR显示脑转移标本KiSS-l mRNA表达仅为原发灶的1／10，与原发灶比较有显著差异（P〈0．01），Western blot检测显示脑转移灶KiSS-l蛋白较原发灶明显减弱，免疫组化显示KiSS-l在原发灶中表达率明显低于原发灶。结论 KiSS-l基因在乳腺痛脑转移中具有转移抑制作用，可能在乳腺癌脑转移治疗中发挥作用。     

BCL-2 family protein expression in initial and recurrent glioblastomas: modulation by radiochemotherapy

OBJECTIVE: In vitro studies indicate a role of apoptosis regulatory proteins of the BCL-2 family in the resistance of glioblastoma multiforme to irradiation and chemotherapy. To date, no study has compared the expression of these proteins in initial and recurrent tumours. The differences of expression of BCL-2, BCL-X, BAX, and MCL-1 proteins of paired first resection and recurrence glioblastoma specimens were examined. METHODS: Immunohistochemistry was performed in 37 cases of glioblastoma multiforme with paraffin embedded tissue from first resections and their recurrences in three treatment groups (15 radiochemotherapy, 15 irradiation, seven untreated). Ten high power fields were evaluated with an arbitrary score (< 5%=1, 5-50%=2, >50%=3), and cumulative scores for each antigen calculated. RESULTS: In the whole group, we found a significant up regulation of antiapoptotic BCL-2 (median cumulative score of 15 in the primary, 19 at recurrence; p<0.0001 in the Wilcoxon test), BCLX (median scores 20 and 25, respectively, p<0.0001), and MCL-1 (median scores 11 and 14, p=0.0395), and a significant down regulation of proapoptotic BAX (median scores 14 and 11, p<0.0001). In the subgroups, these trends were also found. No association between protein expression and treatment regimen was found, although significant changes were restricted to the subgroups that received adjuvant chemotherapy. No significant correlation with clinical prognosis was detected with the Kaplan-Meier method. CONCLUSIONS: In the development from initial to recurrent glioblastoma multiforme, the BCL-2 family rheostat shifts towards antiapoptotic adjustment in vivo. Importantly, the changes in BCL-2 family protein expression characterised here were also seen in the subgroup of patients who did not receive adjuvant radiotherapy or chemotherapy, suggesting that the changes of BCL-2 family protein expression result not only from radiochemotherapy but also reflect the natural course of disease.     

BCL-XL expression levels influence differential regional astrocytic susceptibility to 1,3-dinitrobenzene

The selective vulnerability of brainstem astrocytes to 1,3-dinitrobenzene is mediated by a 10-fold lower threshold for opening of the cyclosporin A-inhibitable mitochondrial permeability transition pore (mtPTP). BCL-XL, BAX and BCL-2 are members of the BCL-2 protein family known to regulate both apoptotic and necrotic cell death signaling at the mtPTP. The levels at which these proteins are expressed relative to one another, where in the cell they are located and whether they are post-translational modified contributes greatly to the balance in active agonistic to active antagonistic BCL-2 proteins, and this critical balance has been hypothesized to dictate regional astrocytic susceptibility to DNB. The effects of DNB on the balance in expression of the BCL-2 family proteins have been evaluated in F344 rat DNB-sensitive (brainstem) and non-sensitive (cortical) tissue homogenates and primary astrocytes. No significant treatment-related alterations in BCL-XL, BAX or BCL-2 protein expression are observed in rat tissue homogenates or primary astrocytes. However, moderate increases in BCL-XL are observed only in DNB-treated rat cortical astrocytes, and these increases may be sufficient to shift the constitutive balance in expression of antagonistic to agonistic BCL-2 proteins from a ratio which favors BAX to one in which BAX and BCL-XL are comparably expressed. Rat primary brainstem and cortical astrocytes are also transiently transfected with bcl-xl to evaluate whether or not moderate enhancement of BCL-XL protein expression levels are sufficient to alter regional sensitivity to DNB in vitro. BCL-XL overexpression minimizes DNB-induced inhibition of succinate dehydrogenase (complex II) activity and increases significantly the concentration of DNB required to induce MPT onset in primary brainstem and cortical astrocytes. Results from the current investigation suggest that modest region-specific alterations in the balance in expression of antagonistic to agonistic BCL-2 proteins may adequately explain differential regional sensitivity to DNB-induced mitochondrial dysfunction.     

Expression of apoptosis-related proteins in model of anoxia in vitro

There has been growing evidence that different modes of cell death exist, among them the apoptosis is thought to be an important mechanism of nerve cell loss implicated in various pathological states. A number of proteins mediated with apoptotic process have been identified, including p53, BAX, BCL-2 and BCL-X. We examined the expression of proteins related to programmed cell death in hippocampal neurons in vitro, exposed to pure anoxia or pretreated with apoptosis modulating agents: zinc and zinc chelator - TPEN. The results evidenced the noticeable differences in the expression of pro- and anti-apoptotic proteins in particular experiments. In the cultures exposed to pure anoxia, a significant increase of p53 and BAX immunoreactivity, associated with the decreased level of BCL-2 and BCL-X immunopositive cells was observed, related to the activation of apoptotic process. Hippocampal cultures pretreated with ZnCl2 before anoxia showed decreased immunoreactivity for p53 and BAX, connected with BCL-2 overexpression, whereas the cultures exposed to zinc chelating agent - TPEN or TPEN connected with anoxia showed significant increase of immunorectivity for p53 and BAX. This strong immunoreactivity of proapototic proteins (p53 and BAX) in hippocampal cultures exposed to anoxia or/and TPEN correlated with previous ultrastructural evidences of anoxia- and TPEN-induced apoptosis, while the overexpression of anti-apoptotic protein (BCL-2 and BCL-X) in zinc-pretreated cultures evidenced the protective ability of this metal against apoptosis in model of anoxia in vitro.     

100例脑肿瘤中p53基因突变与P53蛋白积聚的研究

目的:研究不同类型脑肿瘤中的p53基因突变与P53蛋白积聚及其相关性。方法:采用聚合酶链反应-单链构象多态性(PCR-SSCP)分析及免疫组化法检测100例脑肿瘤p53基因突变及蛋白表达。结果:p53基因突变率为11%(11/100),其中高恶度胶质瘤为37.5%(6/16),低恶度胶质瘤4.3%(1/23),脑膜瘤6.9%(2/29),转移瘤40.0%(2/5)。P53蛋白表达阳性率为22%(22/100),其中高恶度胶质瘤为62.5%(10/16),低恶度胶质瘤为26.1%(6/23),脑膜瘤10.3%(3/29),转移瘤60%(3/5);其他肿瘤均未发现p53基因突变或蛋白表达。P53蛋白表达阳性的22例中伴有p53基因突变者11例,多见于高恶度肿瘤。结论:p53基因失活在脑肿瘤恶性进展过程中起重要作用。p53基因突变与P53蛋白积聚相关,但并非唯一因素。     

Metastatic brain tumor in the elderly

Authors have reviewed 322 consecutive patients with malignant tumors confirmed by pathological studies between October 1973 and August 1987 in order to determine the frequency, clinical presentation, and lesion localization of metastatic brain tumor in the elderly. Among 322 patients with malignant tumor, 7 patients with primary brain tumor and 21 patients with metastatic brain tumors were found. The over-all frequency of metastases to the brain was 5.8%. This frequency of brain metastasis in the elderly was lower than those of the previous literature which have varied from 9 to 35%. The patients' ages with metastatic brain tumor ranged from 65 to 88 years with a median age of 77.5 years. The primary tumor sites of metastatic brain tumors were limited to 5 kinds of organs. These metastases were found in 27.3% of 11 patients with breast cancer, 17.5% of 80 patients with lung cancer, 6.7% of 15 patients with bile duct system cancer, 5.0% of 20 patients with pancreatic cancer, and 2.0% of 91 patients with gastric cancer. There was no brain metastasis in the other kinds of carcinoma. Among 21 metastatic brain tumors, there were 14 patients with lung cancer, 3 patients with breast cancer, 2 patients with gastric cancer, 1 patient with cholangiocarcinoma, and 1 patient with pancreatic cancer. In this series, the frequency of single and multiple metastases were 13 and 8 cases, respectively. The multiple brain metastases ranged from 2 to 6 nodules. In 21 metastatic brain tumors, there were 42 metastatic nodules in total.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metastases of lung cancer to the brain--the examination of angiogenesis and p53 expression

The aim of the present study was to investigate the intensity of angiogenesis and p53 protein expression in metastases of lung cancer to the brain. There were eight cases of squamous cell type and nine adenocarcinomas among 17 examined cases of metastatic carcinomas. The antibodies against von Willebrand factor (vWF)--to highlight the microvessels and against p53 protein--for detection of immunopositive cells were used. The intensity of angiogenesis was represented by the mean number of the blood vessels in three tumor fields with the highest microvascular density ("hot spots"). The measurements were taken in three microscopic fields under 200x magnification (the examined area was 0.785 mm2). The mean number of p53-positive cells in three tumor areas under 200x magnification with the highest number of p53-positive cells was the measure of protein p53 expression. The values of vascular density and p53 expression differed a lot among the examined tumors. The values of vascular density were between 4.2-106 vessels/mm2 (mean value 49.3 vessel/mm2). The number of p53-immunopositive cells was between 0-284 cells/mm2 (mean value 110.6 cells/mm2). There was no significant correlation between examined parameters (correlation coefficient 0.18).     

Comparative study on the expression of stress-response protein (srp) 72, srp27, αB-crystallin and ubiquitin in brain tumours. An immunohistochemical investigation

This immunohistochemical study compares the expression of stress-response (heat-shock) protein (srp) 72, srp 27, αB-crystallin and ubiquitin in 86 primary human brain tumours and 21 carcinoma metastases to the central nervous system. Normal brain tissues were included for control purposes. Serial sections of formalin-fixed, paraffin-embedded tissues were used. Most meningiomas (17/23), glioblastomas (11/12) and breast carcinoma metastases (9/10) and some astrocytomas (7/13), pituitary tumours (4/9) and lung cancer metastases (5/11) had tumour cells that reacted with one or more of the antibodies used. Around 43% of the meningiomas and 25% of the glioblastomas expressed srp 72 only. Sole expression of srp 2 7, αB-crystallin or ubiquitin was seen in several tumours. Some meningiomas (3/23) and breast cancer metastases (4/10) co-expressed srp 72 and srp 27, and 1/3 of the glioblastomas co-expressed srp 27 and αB-crystallin. We conclude that primary and metastatic tumours of the brain produce stress-related proteins and that certain tumours concurrently express two or more srp's.     

硼中子俘获疗法诱导U87胶质瘤细胞凋亡

目的 探讨硼中子俘获疗法(BNCT)对人脑胶质瘤细胞株U87的增殖抑制和诱导凋亡的作用及可能机制.方法 实验分为未照射组(0 Gy)、γ射线对照组(4、8 Gy)、反应堆组(3.5 Gy)、BNCT组(4、8 Gy).采用形态观察、流式细胞仪Annexin V/PI荧光染色、四甲基偶氮唑蓝(MTT)法等方法观察BNCT对U87细胞的增殖抑制和诱导凋亡的作用,以免疫组织化学技术检测P53蛋白的表达,应用western blot检测BCL-2、BAX蛋白表达的变化.结果 硼中子照射后细胞出现典型的凋亡形态改变.BNCT 4、8 Gy组处理后48 h细胞的凋亡率分别为65.1%、85.9%.BNCT 4、8 Gy组细胞生长抑制作用显著高于同等剂量的γ射线照射组(P＜0.01).未照射的U87细胞P53蛋白表达阴性,BNCT4、8 Gy照射后P53蛋白表达阳性.BNCT4、8 Gy照射后BCL-2蛋白表达下降,BAX蛋白上升.结论 BNCT对U87细胞具有显著的增殖抑制作用,并有剂量、时间依赖性特点.     

p53、p16和Rb在原发和继发胶质母细胞瘤中的表达及意义

目的探讨p53、p16和Rb在原发和继发胶质母细胞瘤（GBM）中表达的差异性及其意义。方法对28例原发、32例继发GBM和6例正常脑组织应用免疫组织化学法检测Rb表达，并用RT．PCR和Western blot检测其p53、p16mRNA和蛋白的表达。结果免疫组织化学染色示正常脑组织中无Rb表达，28例原发GBM中有6例表达缺失（21．4％）．32例继发GBM中有4例表达缺失（12．5％），两者相比差异无显著性意义（P〉0．05）。RT．PCR和Western blot检测p53和p16mRNA和蛋白表达，发现所有继发GBM中p53表达强度较原发GBM明显增加（P〈0．051，同时28例原发GBM中有10例p16表达缺失（35．7%），32例继发GBM中仅有2例p16表达缺失（6．25％），两者相比差异有显著性意义（P〈0．01）。结论细胞周期调节基因p53在mRNA和蛋白水平表达增加和p16在同样水平表达缺失是原发与继发GBM重要的细胞周期调控基因的变化，可能是基因治疗GBM的重要靶点。     

Changes of steroidogenic acute regulatory protein mRNA expression in postnatal rat development

The present study investigated the expression pattern of steroidogenic acute regulatory protein (StAR) mRNA in several brain regions and peripheral endocrine organs using Northern blot, RT-PCR and in situ hybridization. StAR mRNA in the adrenal gland was detected at birth and decreased for 2 weeks postnatally. In gonads, it was also detected at birth though at a lower level than adrenal, and was maintained until week 3. Thereafter StAR mRNA expression in both endocrine organs was increased. Though the amount of StAR mRNA in the brain was much less than that of peripheral endocrine organs, it was expressed from birth and, in general, appeared to gradually increase during postnatal development. A gradual increase was found in the hypothalamus, while a sigmoidal expression was shown in the olfactory bulb. The increased expression of StAR mRNA in the postnatal period suggests that it might have a role in the regulation of neurosteroidogenesis needed in neuronal cell growth and differentiation in postnatal rat brain development.     

Expression of stress-response (heat-shock) protein 27 in human brain tumors: an immunohistochemical study

Summary This report concerns the expression of the low molecular weight stress-response (heat-shock) protein 27 (srp 27) in a variety of human brain tumors. Immunohistochemical techniques were used; cells of the breast cancer line MCF7 served as positive controls. The reaction product was found exclusively in the cytoplasm. Srp 27 was detected in 5/5 breast tumor metastases to the brain and in 5/21 meningiomas. The protein was also detected in 5/11 glioblastomas and 2/5 pituitary adenomas. By comparison, positive staining was observed in only 1/15 astrocytomas and 1/7 medulloblastoma and no reaction was seen with the oligodendrogliomas, schwannomas and gangliogliomas tested. These observations demonstrate that srp 27 is expressed by certain primary intracranial tumors.     

Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in human brain tumors: relationships of iNOS to superoxide dismutase (SOD) proteins (SOD1 and SOD2), Ki-67 antigen (MIB-1) and p53 protein

In this study, inducible nitric oxide synthase (iNOS) expression in a series of 158 human primary brain tumors was analyzed. To gain some insight into the biological significance of iNOS expression in tumor cells, comparative immunohistochemical analyses were employed to characterize the expression of iNOS, superoxide dismutase (SOD) proteins (SOD1 and SOD2), Ki-67 antigen (MIB-1) and p53 protein in these cells. Sixteen (39.0%) of the 41 glioblastoma multiforme (GBM) specimens showed iNOS immunoreactivity. Positive immunoreactions with iNOS were also detected in 2/8 anaplastic astrocytomas, 1/17 astrocytomas, 1/14 medulloblastomas and 1/11 primitive neuroectodermal tumors, but no positive reactions were observed in oligodendrogliomas (0/11), ependymomas (0/5), schwannomas (0/21), meningiomas (0/23) or pituitary adenomas (0/7). The MIB-1 labeling index of GBMs that expressed iNOS was significantly higher than that of GBMs that did not (0.025< P <0.05, Wilcoxon rank-sum test). Unlike iNOS-negative tumors, all iNOS-positive tumors coexpressed SOD1 or SOD2. In particular, there was a significant correlation between iNOS induction and SOD1 expression (P =1.65x10(-10), Fisher's exact test) in GBM specimens. There was no significant relationship between iNOS and p53 protein in any type of primary brain tumor (P >0.05, Fisher's exact test). No significant immunohistochemical reactions with iNOS, MIB-1 or p53 protein were observed in normal brain tissue sections. We conclude that primary brain tumors express iNOS, and that iNOS expression in brain tumor cells may depend, in part, on cellular proliferation potential. Based on the fact that SOD1 scavenges oxidative-stress species originating from large amounts of nitric oxide (NO) produced by iNOS, iNOS-expressing brain tumor cells may protect themselves against NO cytotoxicity by overinducing SOD1.     

脑肿瘤中P53蛋白的表达及Ki－67LI的相关性研究

利用突变型Ｐ５３蛋白的单抗及Ｋｉ－６７单抗对５０例冰冻标本进行检测，结果发现，脑肿瘤中突变型Ｐ５３蛋白的表达阳性率为４６％。低恶度胶质瘤、高恶度胶质瘤及转移癌中Ｐ５３蛋白表达的阳性率不同，分别为１８．２％、５３．８％及１００％。Ｐ５３蛋白表达阳性的肿瘤中高Ｋｉ－６７ＬＩ比例为７０％，其中位数及均数为１４．３％、２０．７６±１８．３（％），而无Ｐ５３蛋白表达的脑瘤中，ｋｉ－６７ＬＩ中位数及均数分别为１．３９及５．１９±９．０（％）。结果表明，突变型Ｐ５３蛋白的表达与脑肿瘤的组织类型，分化程度及细胞的增殖有关，而它的高表达又可能是肿瘤恶性表型或转移的标志之一。     

Steroidogenic acute regulatory protein expression in the normal human brain and intracranial tumors

In this study we found the expression of steroidogenic acute regulatory protein (StAR) mRNA and protein in both the normal human brain and intracranial tumors, using RT-PCR and immunohistochemistry. StAR mRNA is expressed at a very low level in the white matter of the normal human brain, but in tumoral tissues StAR mRNA was specifically expressed in oligodendrogliomas and malignant glial tumors. StAR-positive cells were also detected in the normal human brains and gliomas; its frequency and density were higher in glioma tissue. These findings suggest that StAR expression might be correlated with the growth of glial tumors.