Antibody-mediated p53 protein therapy prevents liver metastasis in vivo

To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated p53 protein therapy, an Fv-p53 fusion protein produced in Pichia pastoris was tested on CT26.CL25 colon cancer cells in vitro and in vivo in a mouse model of colon cancer metastasis to the liver. In vitro experiments showed killing of CT26.CL25 cells by Fv-p53 but not Fv or p53 alone, and immunohistochemical staining confirmed that Fv was required for transport of p53 into cells. Prevention of liver metastasis in vivo was tested by splenic injection of 100 nmol/L Fv-p53 10 min and 1 week after injection of CT26.CL25 cancer cells into the portal vein of BALB/c mice. Mice were sacrificed 1 week after the second injection of Fv-p53 and assigned a quantitative metastasis score. Control mice had an average metastasis score of 3.3 +/- 1.3, whereas mice treated with Fv-p53 had an average metastasis score of 0.8 +/- 0.4 (P = 0.004). These results indicate that Fv-p53 treatment had a profound effect on liver metastasis and represent the first demonstration of effective full-length p53 protein therapy in vivo. mAb 3E10 Fv has significant clinical potential as a mediator of intracellular and intranuclear delivery of p53 for prevention and treatment of cancer metastasis.     

大肠癌原发灶和肝转移灶p53变异及蛋白表达

目的 了解Ｐ５３及其蛋白在大肠癌发生、转移过程中的动态变化。方法Ｐ５３基因外显子５－９以ＤＧＧＥ及自动ＤＮＡ序列分析来检测,Ｐ５３蛋白表达以免疫组化方法检测。结果 ３４例中２１例呈Ｐ５３变异,占６１．８％,其中５例仅在肝转移灶发现Ｐ５３变异,其余均为原发灶、转移灶生的变异。另有２例原发灶即有Ｐ５３变异者,在转移灶出现了新增加的变异。在３７处变异中,２７和为错义突变占７３．０％;６处为无义突变占１６     

软组织肉瘤中p53基因的突变和蛋白的表达研究

目的检测软组织肉瘤中p53基因的突变和蛋白的表达情况。方法应用PCR-SSCP和DNA测序的方法检测p53基因的突变,免疫组织化学技术Envision二步法检测p53蛋白的表达。结果 63例软组织肉瘤中检测到p53基因突变18例（28.6%）,突变位点主要在第6外显子和第7外显子,突变和组织学分级间无明显相关性;63例p53蛋白的表达44例,蛋白的表达与组织学分级间无明显相关性;且p53基因的突变与p53蛋白的表达无明显相关性。结论 p53基因的突变和p53蛋白的表达可能作为软组织肉瘤发生的一个重要因素。     

p53 protein expression by hepatocarcinogens in the rat liver and its potential role in mitoinhibition of normal hepatocytes as a mechanism of hepatic tumour promotion

The tumour suppressor gene p53 is expressed in response to DNA-damage; its protein product blocks cells in the G1-phase of the cell cycle. This gives cells additional time to repair their DNA-damage. However, it may trigger apoptosis if damage is too high. Loss of p53 function appears to be an important step in carcinogenesis because 50% of human tumours have lost functional p53. In order to study the role of p53 in experimental hepatocarcinogenesis, we determined the expression of p53 in rat liver in response to various hepatocarcinogenic and hepatotoxic compounds. Administration of hepatocarcinogenic compounds increased p53 protein levels in the liver as detected by immunoprecipitation followed by SDS-PAGE and Western blotting with ECL-detection. The hepatocarcinogens included N-hydroxy-2-acetylaminofluorene, aflatoxin B1, and diethylnitrosamine. Their structural analogues N-hydroxy-4- acetylaminobiphenyl and ethyl methane-sulphonate which are not hepatocarcinogenic, did not induce p53. Also, two hepatotoxic compounds (carbon tetrachloride, D-galactosamine) did not induce p53. Other compounds that induced p53 in the rat liver were 2-aminofluorene (administered by drinking water for two weeks) and tris-(2,3- dibromopropyl)phosphate. Benzo[a]pyrene did not induce p53. N-Hydroxy-2- acetylaminofluorene, aflatoxin B1, and diethylnitrosamine are potent hepatic tumour promoters. At the same time, they induce p53 protein expression and inhibit proliferation of normal hepatocytes. Because this is not observed with non-hepatocarcinogenic analogues, it suggests an involvement of p53 expression in hepatic tumour promotion. A possible mechanism is discussed.      

Lack of p53 protein expression in preneoplastic rat hepatocytes in vitro after exposure to N-acetoxy-acetylaminofluorene, X-rays or a proteasome inhibitor

Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays.     

p53/bcl-2与放疗诱导的宫颈癌细胞凋亡关系的研究

目的探讨宫颈癌放疗过程中,放疗诱导的细胞凋亡与p53、bcl-2的相关性.方法选择未经治疗的宫颈癌病人20例为实验对象,搜集分割放疗前后宫颈癌组织标本,用TDT-mediated dUTP-biotin nick end labeling(TUNEL)方法检测凋亡细胞;采用单克隆抗体免疫组化ABC法检测细胞凋亡相关基因p53、bcl-2的蛋白表达水平.结果(1)在宫颈癌放疗前后,细胞凋亡阳性率和平均凋亡指数分别为25%和0.11%、75%和2.8%,放疗前后有显著差异(P＜0.001);(2)放疗后p53蛋白表达显著减少,bcl-2蛋白无显著变化;(3)放疗前后,p53基因的表达与细胞凋亡呈有意义的相关性变化.结论放射治疗诱导了宫颈癌细胞凋亡的发生,并与凋亡调节基因p53及其表达呈间接有关.     

Expression of the novel tumour suppressor p33(ING1)is independent of p53

A recently cloned tumour suppressor candidate, p33ING1, has been shown in vitro to collaborate with p53 to execute growth arrest and apoptosis. However, it is unclear as to how the expression of ING1 is regulated in normal and stress conditions. Using a p53-knockout mouse model, we investigated if the expression of ING1 was dependent on p53. We found that there was no difference in ING1 mRNA and protein levels between p53+/+ and p53-/- murine organs. In addition, when normal human epithelial keratinocytes (NHEK) and a keratinocyte cell line, HaCaT, which lacks wild-type p53 function, were exposed to UVB irradiation, the expression levels of ING1 were elevated in both NHEK and HaCaT cells. It is interesting, however, that UVB irradiation did not induce ING1 expression in dermal fibroblasts isolated from p53+/+ and p53-/- mice. Based on our findings, we therefore conclude that the expression of ING1 is independent of p53 status. UV induction of ING1 in keratinocytes suggests that ING1 may play a role in cellular stress response and skin carcinogenesis.     

Tissue Microarray Immunohistochemical Profiles of p53 and pRB in Hepatocellular Carcinoma and Hepatoblastoma

The tumour suppressor genes, p53 and pRb, are known to play important roles in neoplastic transformation.While molecular routes to the uncontrolled growth of hepatocytes, leading to primary liver cancer have generatedconsiderable interest, the roles of p53 and pRb mutations in hepatocellular carcinoma (HCC) and hepatoblastoma(HB) remain to be clarified. We examined the immunohistochemical expression of p53 and pRb gene products in26 HCC and 9 HB, sampled into tissue microarray blocks. 10 (38%) of 26 HCC showed > 10% tumour nuclearstaining for p53 protein, 3 of these also being HbsAg positive. Conversely, none of 9 HB expressed nuclear p53immunopositivity. Some 24 (92%) HCC and 8 (89%) HB showed loss of pRb nuclear expression. Two of the 26HCC and one of the 9 HB showed >10% tumour nuclear staining for pRb protein. Our results suggest that p53does not have an important role in the development of HB but may contribute in HCC. There is also loss of pRbexpression in the majority of HCC and HB, supporting loss of pRb gene function in the hepatocarcinogenesispathway. However, a comparison of the staining profiles of p53 and pRb proteins in HCC and HB did not reveala consistent pattern to differentiate between the two types of tumours immunohistochemically. Hence the useof p53 and pRB protein expression has no contribution in the situation where there is a diagnostic difficulty indeciding between HCC and HB.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts and increased p53 protein in mouse skin

p53 protein expression has been shown to increase in responseto DNA damage in cell culture systems. We have Studied p53 expressionand benzo[a]pyrene (B[a]P) -induced DNA-damage in the form ofbenzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts as measuredby synchronous fluorescence spectrophotometry (SFS) in B[a]P-treatedC57BL/6 mouse skin. Polyclonal murine antibody CM5, which iscomparable to human CM1, detecting both wild-type and mutatedprotein, was used. BPDE-DNA adducts reached their maximum at24 h after all dosage regimens, but were very well detectablealso at 12 and 48 h after the treatment, while no adducts weremeasurable at 1 week and thereafter. P53 expression was seenin 9/17(53%) skin samples from mouse treated with 500 µgof B[a]P 12–48 h after the treatment, while all 25 (100%)cases of similarly treated mouse skins were negative after 30weeks of the treatment. Only one positive sample of total 11was found among mice treated with repeated 62.5 µg dosesand this was 24 h after the last treatment. After one 62.5 µgdose all mice were negative. This is the first report of anassociation of p53 protein with DNA damage in vivo and givessupport for the putative function of p53 in cellular defensemachinery towards chemical damage.     

Cleavage of poly(ADP-ribose) transferase during p53-independent apoptosis in rat liver after treatment with N-nitrosomorpholine and cyproterone acetate.

The aim of this work was to study the role of the tumor suppressor p53 and of poly(ADP-ribose) transferase (pADPRT) in the control of hepatocyte apoptosis in two different in vivo models, i.e., during the process of tumor initiation by the genotoxin and cytotoxin N-nitrosomorpholine (NNM) and after withdrawal of the hepatomitogen cyproterone acetate (CPA). Treatment with NNM induces apoptosis followed by necrosis and regenerative DNA synthesis. At the first wave of apoptosis 12 h after NNM application, no p53 expression could be detected by immunohistochemical analysis and immunoblotting. However, 24 h after treatment, numerous p53-positive hepatocyte nuclei were detected, whereas hepatocytes in early and later stages of apoptosis were always negative. Simultaneously with the increased p53 levels, p21 protein was induced. This was accompanied by a block in replicative DNA synthesis, as detected by proliferating-cell nuclear antigen immunostaining. Concomitantly with the increase in apoptosis, dramatic degradation of the nuclear enzyme pADPRT was observed, as evidenced by immunoblotting and activity blotting. The decrease in pADPRT enzymatic activity observed 12 h after treatment coincided with the greatest extent of pADPRT cleavage. One prominent cleavage product was 64 kDa, suggesting that granzyme B was involved in pADPRT degradation. In the second in vivo model we used, i.e., withdrawal of treatment with the hepatomitogen CPA, apoptosis of excessive hepatocytes but no necrosis occurs. Again, no induction of p53 expression could be detected in the liver even at the maximum level of apoptosis, whereas a strong correlation between induction of apoptosis and cleavage of pADPRT to a 64-kDa fragment was observed. These results from whole-animal experiments strongly suggest that the induction of apoptosis in rat liver after genotoxic and cytotoxic damage and during regression of hyperplasia is driven by a p53-independent pathway but is accompanied by cleavage of pADPRT.     

p53 Mutations and protein overexpression in primary colorectal cancer and its liver metastasis

Although there have already been many studiesreported about p53 statIJs on primary colorectal cancerand / or hepatic metastases,t'. 2] we have not found anyrepoft that compares p53 status between primary andmetastatic lesions in each patient on an individual level.34 patieflts with colorectal cancer and liver metastaseswere chosen for this study. P53 status in Primary and livermetastatic tllmor lesions of every individual wereinvestigated, in order to understand that p53 gene statusduring tumo…     

Pokemon和p53蛋白在肝细胞癌组织中的表达及临床意义

目的：探讨Pokemon和p53蛋白在人肝细胞癌组织中的表达,分析其与肝细胞癌转移复发的关系。方法：应用免疫组织化学法检测60例肝细胞癌组织和癌旁组织中Pokemon和p53蛋白的表达情况。结果：60例肝细胞癌组织中Pokemon和p53表达率显著高于癌旁组织（P〈0.05）。Pokemon的表达与临床分期、肿瘤直径、肿瘤分化程度密切相关（P〈0.05）;p53的表达与脉管癌栓、术后复发、肝外转移及肿瘤分化程度密切相关（P〈0.05）。Pokemon和p53的表达呈显著正相关（γ=-0.275,P=0.033）。结论：Pokemon和p53蛋白的高表达可能在肝细胞癌的发生及进展中起着重要作用。     

P53蛋白在胰腺癌与癌旁组织中表达的临床意义

目的：了解胰腺癌及其癌旁组织的ｐ５３蛋白表达，并分析其临床意义。方法：作者对３６例胰腺导管癌、２５例胰腺癌旁组织、１０例慢性胰腺炎和１２例正常胰腺组织石蜡标本进行ｐ５３单抗的免疫组化染色。结果：３６例胰腺癌和２５例癌旁组织中呈ｐ５３蛋白阳性染色分别为２１例（５８％）和１１例（４４％），且其阳性率间有显著相关性。（Ｐ＜０．０１）。而慢性胰腺炎和正常胰腺组织均未见ｐ５３蛋白阳性染色，与胰腺癌和癌旁组织ｐ５３阳性率间均有显著差异（Ｐ＜０．０１）。不同临床分期及组织学分型胰腺癌，ｐ５３蛋白阳性染色率间无显著差异。结论：ｐ５３蛋白的表达与胰腺癌的发生有关；胰腺癌旁组织中某些细胞呈ｐ５３阳性染色，这些细胞可能较易发生癌变，使胰腺癌呈多中心性发生。     

p53 protein over-expression and p53 gene abnormalities in HIV-1-related non-Hodgkin's lymphomas

Alteration of the p53 tumor-suppressor gene was studied in non-Hodgkin's lymphomas (NHLs) from HIV-I-infected patients. p53 protein was over-expressed in 10 out of the 45 (22%) cases analyzed, mainly clustering in the small-non-cleaved-cell (SNC) (5/19) and Ki-l⁺ anaplastic large-cell (ALC) (3/8) subtypes, according to previous findings on HIV-1 -unrelated NHLs. p53-positive small-non-cleaved-cell lymphomas presented a diffuse or clustered pattern of p53-positive neoplastic cells consequent upon p53-gene mutations. In contrast, in Ki-1 + ALC lymphomas p53 immunohistochemical reactivity was limited to scattered tumor cells, and no p53-gene alterations could be detected. These results suggest that p53-gene alterations play a role in the lymphomagenetic process of a fraction of HIV-I-related SNC NHLs, however with a frequency no different from that observed in HIV-I -unrelated NHLs of the same sub-type. In HIV-I-related Ki-I+ ALC lymphomas, mechanisms different from gene alterations might be implicated in over-expression of p53 protein.     

Alterations and prognostic significance of p16 and p53 protein expression in subgroups of cutaneous melanoma

The role of p16 and p53 alterations in cutaneous melanoma has been recently discussed, but it remains to be clarified. In the present immunohistochemical study, the expression of p16 and p53 proteins and their possible prognostic relevance have been examined in 102 melanomas of the aggressive nodular type. Twelve percent showed a strong expression of p53 protein, and these cases were significantly more frequent in the head/neck area compared with other sites (32% vs. 6%). Expression of p16 protein was negative or weak in 9% of the cases, and this tended to be less frequent in head/neck tumors compared with the others (0% vs. 12%). Whereas p53 staining was not prognostically important, loss of p16 staining was significantly associated with markedly reduced recurrence free and patient survival in univariate analysis (product-limit method). In multivariate analysis, lack of p16 staining was significantly associated with recurrent disease (p = 0.013). Our findings indicate an important role of altered p16 protein expression in a subgroup of melanoma patients. Int. J. Cancer 74:535–539, 1997. © 1997 Wiley-Liss, Inc.     

乳腺浸润性导管癌组织中 HPV18 DNA 及 p53 蛋白的表达

目的：探讨人乳腺浸润性导管癌组织中HPV18DNA和p53蛋白的表达及二者间的关系。方法：采用分子原位杂交和免疫组化技术检测60例乳腺浸润性导管癌、30例乳腺腺病和30例正常乳腺组织中HPV18DNA和p53蛋白。结果：癌组中HPV18DNA阳性和HPV18DNA阴性两组间p53蛋白的阳性表达均有显著性差异（P〈0．03），HPV18DNA阳性组与p53蛋白阳性表达呈显著负相关（r=-0．2954、P〈0．04）。结论：HPV18感染后导致野生型p53的灭活和降解可能涉及HPV18感染后人乳腺上皮细胞癌变的转化过程。     

p53和p21基因蛋白表达与胃癌侵袭力的关系

采用免疫组织化学方法研究了６８例胃癌中癌细胞ｐ５３和ｐ２１基因蛋白表达与癌细胞侵袭力的关系。结果表明：６８例胃癌中有３６例ｐ５３基因蛋白染色阳性（５２．９％）,４８例ｐ２１基因蛋白染色阳性（７０．６％）;浸润于浆膜层和肌层的癌细胞ｐ５３蛋白染色的阳性程度明显高于粘膜层癌细胞（Ｐ＜０．０５）;浸润性生长的癌细胞中ｐ５３和ｐ２１蛋白阳性程度均明显强于膨胀性生长的癌细胞（Ｐ＜０．０５）;淋巴结转移病例的癌细胞其ｐ５３和ｐ２１蛋白染色阳性率（分别为５４．３％和７３．９％）与无淋巴结转移的病例（分别为５０．０％和６３．６％）无显著性差异（Ｐ＞０．０５）;有淋巴结转移的病例中,原发癌ｐ５３蛋白阳性强度与转移癌正相关（γ＝０．６８,Ｐ＜０．０１）。结果提示：ｐ５３和ｐ２１基因蛋白染色阳性程度较高的胃癌细胞具有较强的侵袭力。     

Usefulness of p53 protein, Bcl-2 protein and Ki-67 as predictors of chemosensitivity of malignant tumors

In various types of human malignant tumors, the presence or absence of expression of apoptosis-associated gene products (p53 protein and Bcl-2 protein) and the tumor proliferation activity-related factor (Ki-67) was assessed by immunohistochemical staining and the correlation between this expression and chemosensitivity to anticancer drugs was investigated. Study subjects comprised 55 preoperative patients with untreated malignant tumors (9 with esophageal cancer, 11 with stomach cancer, 11 with colon cancer, 13 with hepatic cancer and 11 with breast cancer). A chemosensitivity test was carried out with the histoculture drug response assay (HDRA) method using 4 drugs, mitomycin C (MMC), 5-fluorouracil (5-FU), doxorubicin hydrochloride (ADM), and cisplatin (CDDP). Immunohistochemical staining was used to assess expression of p53 protein, Bcl-2 protein and Ki-67. The tumor growth inhibition index (I.I.) of the 4 drugs was significantly lower in a group of the patients with p53 protein overexpression-type (mutant p53 protein positive expression-type) tumors than in a group with p53 protein negative expression-type tumors (p<0.05). No significant correlation was found between the expression of the Bcl-2 protein by and the I.I. of any drug studied in any type of cancer. A negative correlation was found between the labeling index (L.I.) for Ki-67 in all cases and I.I. for MMC and ADM and thus, chemosensitivity of the tumors with high growth activity was lower. Furthermore, a positive correlation existed between the L.I. for Ki-67 and that for p53 protein. The patients with p53 protein overexpression-type (mutant p53 protein positive) tumors showed low chemosensitivity. In addition, overexpression of p53 protein is suggested to be one of the factors involved in the lowered chemosensitivity of the tumors with high growth activity. Summarizing these findings, the p53 protein can play an important role in cancer therapy.