Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates.

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10-11-10-8 M) against a variety of HER2-expressing cancer cell lines. Several peptide-linked variants of the lead compound were prepared and conjugated to trastuzumab to afford ADCs with drug-to-antibody (DAR) ratios ranging from 3 to 5. The ADCs exhibited antigen-dependent cytotoxicity in vitro and potently suppressed tumor xenograft growth in vivo in a target-dependent manner. Moreover, the ADCs were well-tolerated in both mouse and rat. This work demonstrates for the first time that PBD polyamide hybrids can serve as effective ADC payloads.

Orphan nuclear receptor TR3/Nur77 biologics inhibit tumor growth by targeting angiogenesis and tumor cells.

Pathological angiogenesis is a hallmark of many diseases. Previously, we reported that orphan nuclear receptor TR3/Nur77 was a critical mediator of angiogenesis to regulate tumor growth, sepsis and skin wound healing. However, none of the TR3/Nur77 targeting molecule has been in clinical trial so far. Here, we designed and generated novel TR3 shRNAs and two minigenes that had therapeutic potential for cancer treatment. In addition to extend our previous findings that tumor growth was inhibited in Nur77 knockout mice, we found that metastasis of colorectal tumor was completely inhibited in Nur77-/- mice. Tumor masses were increased ~70% and decreased ~40% in our transgenic EC-Nur77-S mice and EC-Nur77-DN mice, in which the full-length cDNA and the dominant negative mutant of TR3/Nur77 were inducibly and specifically expressed in mouse endothelium, respectively. TR3 was highly expressed in the vasculature and tumor cells of human melanoma and colorectal cancer tissues, but not in normal tissues. The novel TR3 shRNAs and two minigenes almost completely inhibited the proliferation and migration of HUVECs and human melanoma A375sm cells. Angiogenesis induced by adenoviruses expressing VEGF and melanoma growth in mice were greatly and significantly inhibited by systemically administration of adenoviruses expressing TR3 shRNAs and two minigenes. Tumor angiogenesis and the expressions of genes associated with angiogenesis were greatly regulated in tumor tissues treated with TR3 shRNAs and minigenes. Taken together, these studies demonstrated that TR3/Nur77 was a specific therapeutic target for several human cancers by targeting both tumor cells and tumor microenvironment. These TR3/Nur77 biologics inhibit angiogenesis and tumor growth, and have translational potential.

Orphan nuclear receptor TR3/Nur77 improves wound healing by upregulating the expression of integrin ß4.

Tissue repair/wound healing, in which angiogenesis plays an important role, is a critical step in many diseases including chronic wound, myocardial infarction, stroke, cancer, and inflammation. Recently, we were the first to report that orphan nuclear receptor TR3/Nur77 is a critical mediator of angiogenesis and its associated microvessel permeability. Tumor growth and angiogenesis induced by VEGF-A, histamine, and serotonin are almost completely inhibited in Nur77 knockout mice. However, it is not known whether TR3/Nur77 plays any roles in wound healing. In these studies, skin wound-healing assay was performed in 3 types of genetically modified mice having various Nur77 activities. We found that ectopic induction of Nur77 in endothelial cells of mice is sufficient to improve skin wound healing. Although skin wound healing in Nur77 knockout mice is comparable to the wild-type control mice, the process is significantly delayed in the EC-Nur77-DN mice, in which a dominant negative Nur77 mutant is inducibly and specifically expressed in mouse endothelial cells. By a loss-of-function assay, we elucidate a novel feed-forward signaling pathway, integrin ß4 → PI3K → Akt → FAK, by which TR3 mediates HUVEC migration. Furthermore, TR3/Nur77 regulates the expression of integrin ß4 by targeting its promoter activity. In conclusion, expression of TR3/Nur77 improves wound healing by targeting integrin ß4. TR3/Nur77 is a potential candidate for proangiogenic therapy. The results further suggest that TR3/Nur77 is required for pathologic angiogenesis but not for developmental/physiologic angiogenesis and that Nur77 and its family members play a redundant role in normal skin wound healing.

The vascular permeabilizing factors histamine and serotonin induce angiogenesis through TR3/Nur77 and subsequently truncate it through thrombospondin-1.

Angiogenesis plays an important role in cancer and in many other human diseases. Vascular endothelial growth factor-A (VEGF-A), the best known angiogenic factor, was originally discovered as a potent vascular permeability factor (VPF), suggesting that other vascular permeabilizing agents, such as histamine and serotonin, might also have angiogenic activity. We recently demonstrated that, like VEGF-A, histamine and serotonin up-regulate the orphan nuclear receptor and transcription factor TR3 (mouse homolog Nur77) and that TR3/Nur77 is essential for their vascular permeabilizing activities. We now report that histamine and serotonin are also angiogenic factors that, at low micromolar concentrations, induce endothelial cell proliferation, migration and tube formation in vitro, and angiogenesis in vivo. All of these responses are mediated through specific histamine and serotonin receptors, are independent of VEGF-A, and are directly dependent on TR3/Nur77. Initially, the angiogenic response closely resembled that induced by VEGF-A, with generation of "mother" vessels. However, after ~10 days, mother vessels began to regress as histamine and serotonin, unlike VEGF-A, up-regulated the potent angiogenesis inhibitor thrombospondin-1, thereby triggering a negative feedback loop. Thus, histamine and serotonin induce an angiogenic response that fits the time scale of acute inflammation.

Orphan nuclear transcription factor TR3/Nur77 regulates microvessel permeability by targeting endothelial nitric oxide synthase and destabilizing endothelial junctions.

Low-level basal vascular permeability (BVP) provides nutrients to normal tissues, and increased vascular permeability is characteristic of inflammation and cancer. We recently reported that VEGF-A, a potent vascular permeabilizing and angiogenic factor, exerts much of its angiogenic activity by up-regulating expression of TR3/Nur77, an orphan nuclear transcription factor, in vascular endothelial cells (EC). To determine whether TR3/Nur77 had a more general role in regulating vascular permeability, we found that histamine, serotonin, and platelet-activating factor, small molecule vascular permeabilizing agents, also increased TR3/Nur77 expression acutely in EC. BVP and the acute vascular hyperpermeability (AVH) induced by these vascular permeabilizing factors were greatly decreased in Nur77(-/-) mice, and both BVP and AVH correlated with Nur77 expression levels in several different mouse strains. BVP and AVH were enhanced in transgenic mice in which Nur77 was selectively overexpressed in vascular EC, whereas both were suppressed in mice overexpressing dominant-negative Nur77. Chronic vascular hyperpermeability (CVH) was induced long before the onset of angiogenesis in a modified, in vivo Matrigel assay that included PT67 cells packaging retroviruses expressing Nur77-sense, whereas inclusion of cells packaging viruses expressing Nur77-antisense prevented VEGF-A-induced CVH. TR3/Nur77 modulated vascular permeability by increasing endothelial nitric-oxide synthase expression and by downregulating several EC junction proteins that maintain vascular homeostasis. Both functions required TR3/Nur77 transcriptional activity. Taking these data together, TR3/Nur77 is up-regulated by several vascular permeabilizing agents and has critical roles in mediating BVP, AVH, and CVH.

Development of a Novel DNA Mono-alkylator Platform for Antibody-Drug Conjugates.

Although microtubule inhibitors (MTI) remain a therapeutically valuable payload option for antibody-drug conjugates (ADC), some cancers do not respond to MTI-based ADCs. Efforts to fill this therapeutic gap have led to a recent expansion of the ADC payload "toolbox" to include payloads with novel mechanisms of action such as topoisomerase inhibition and DNA cross-linking. We present here the development of a novel DNA mono-alkylator ADC platform that exhibits sustained tumor growth suppression at single doses in MTI-resistant tumors and is well tolerated in the rat upon repeat dosing. A phosphoramidate prodrug of the payload enables low ADC aggregation even at drug-to-antibody ratios of 5:1 while still delivering a bystander-capable payload that is effective in multidrug resistant (MDR)-overexpressing cell lines. The platform was comparable in xenograft studies to the clinical benchmark DNA mono-alkylator ADC platform DGN459 but with a significantly better tolerability profile in rats. Thus, the activity and tolerability profile of this new platform make it a viable option for the development of ADCs.

Site-Specific Dolasynthen Antibody-Drug Conjugates Exhibit Consistent Pharmacokinetic Profiles across a Wide Range of Drug-to-Antibody Ratios.

Key defining attributes of an antibody-drug conjugate (ADC) include the choice of the targeting antibody, linker, payload, and the drug-to-antibody ratio (DAR). Historically, most ADC platforms have used the same DAR for all targets, regardless of target characteristics. However, recent studies and modeling suggest that the optimal DAR can depend on target expression level and intratumoral heterogeneity, target internalization and trafficking, and characteristics of the linker and payload. An ADC platform that enables DAR optimization could improve the success rate of clinical candidates. Here we report a systematic exploration of DAR across a wide range, by combining THIOMAB protein engineering technology with Dolasynthen, an auristatin-based platform with monomeric and trimeric variants. This approach enabled the generation of homogeneous, site-specific ADCs spanning a discrete range of DARs 2, 4, 6, 12, and 18 by conjugation of trastuzumab IgG1 THIOMAB constructs with 1, 2, or 3 engineered cysteines to monomeric or trimeric Dolasynthen. All ADCs had physicochemical properties that translated to excellent in vivo pharmacology. Following a single dose of ADCs in a HER2 xenograft model with moderate antigen expression, our data demonstrated comparable pharmacokinetics for the conjugates across all DARs and dose-dependent efficacy of all test articles. These results demonstrate that the Dolasynthen platform enables the generation of ADCs with a broad range of DAR values and with comparable physiochemical, pharmacologic, and pharmacokinetics profiles; thus, the Dolasynthen platform enables the empirical determination of the optimal DAR for a clinical candidate for a given target.

Discovery and Preclinical Characterization of XMT-1660, an Optimized B7-H4-Targeted Antibody-Drug Conjugate for the Treatment of Cancer.

Antibody-drug conjugates (ADC) achieve targeted drug delivery to a tumor and have demonstrated clinical success in many tumor types. The activity and safety profile of an ADC depends on its construction: antibody, payload, linker, and conjugation method, as well as the number of payload drugs per antibody [drug-to-antibody ratio (DAR)]. To allow for ADC optimization for a given target antigen, we developed Dolasynthen (DS), a novel ADC platform based on the payload auristatin hydroxypropylamide, that enables precise DAR-ranging and site-specific conjugation. We used the new platform to optimize an ADC that targets B7-H4 (VTCN1), an immune-suppressive protein that is overexpressed in breast, ovarian, and endometrial cancers. XMT-1660 is a site-specific DS DAR 6 ADC that induced complete tumor regressions in xenograft models of breast and ovarian cancer as well as in a syngeneic breast cancer model that is refractory to PD-1 immune checkpoint inhibition. In a panel of 28 breast cancer PDXs, XMT-1660 demonstrated activity that correlated with B7-H4 expression. XMT-1660 has recently entered clinical development in a phase I study (NCT05377996) in patients with cancer.

Orphan nuclear receptor TR3/Nur77 regulates VEGF-A-induced angiogenesis through its transcriptional activity.

Vascular endothelial growth factor (VEGF)-A has essential roles in vasculogenesis and angiogenesis, but the downstream steps and mechanisms by which human VEGF-A acts are incompletely understood. We report here that human VEGF-A exerts much of its angiogenic activity by up-regulating the expression of TR3 (mouse homologue Nur77), an immediate-early response gene and orphan nuclear receptor transcription factor previously implicated in tumor cell, lymphocyte, and neuronal growth and apoptosis. Overexpression of TR3 in human umbilical vein endothelial cells (HUVECs) resulted in VEGF-A-independent proliferation, survival, and induction of several cell cycle genes, whereas expression of antisense TR3 abrogated the response to VEGF-A in these assays and also inhibited tube formation. Nur77 was highly expressed in several types of VEGF-A-dependent pathological angiogenesis in vivo. Also, using a novel endothelial cell-selective retroviral targeting system, overexpression of Nur77 DNA potently induced angiogenesis in the absence of exogenous VEGF-A, whereas Nur77 antisense strongly inhibited VEGF-A-induced angiogenesis. B16F1 melanoma growth and angiogenesis were greatly inhibited in Nur77-/- mice. Mechanistic studies with TR3/Nur77 mutants revealed that TR3/Nur77 exerted most of its effects on cultured HUVECs and its pro-angiogenic effects in vivo, through its transactivation and DNA binding domains (i.e., through transcriptional activity).

Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

The Dolaflexin-based Antibody-Drug Conjugate XMT-1536 Targets the Solid Tumor Lineage Antigen SLC34A2/NaPi2b.

Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.

Down syndrome candidate region 1 isoform 1 mediates angiogenesis through the calcineurin-NFAT pathway.

Down syndrome candidate region 1 (DSCR1) is one of more than 50 genes located in a region of chromosome 21 that has been implicated in Down syndrome. DSCR1 can be expressed as four isoforms, one of which, isoform 4 (DSCR1-4), has recently been found to be strongly induced by vascular endothelial growth factor A (VEGF-A(165)) and to provide a negative feedback loop that inhibits VEGF-A(165)-induced endothelial cell proliferation in vitro and angiogenesis in vivo. We report here that another DSCR1 isoform, DSCR1-1L, is also up-regulated by VEGF-A(165) in cultured endothelial cells and is strongly expressed in several types of pathologic angiogenesis in vivo. In contrast to DSCR1-4, the overexpression of DSCR1-1L induced the proliferation and activation of the transcription factor NFAT in cultured endothelial cells and promoted angiogenesis in Matrigel assays in vivo, even in the absence of VEGF-A. Similarly, small interfering RNAs specific for DSCR1-1L and DSCR1-4 had opposing inhibitory and stimulatory effects, respectively, on these same functions. DSCR1-4 is thought to inhibit angiogenesis by inactivating calcineurin, thereby preventing activation and nuclear translocation of NFAT, a key transcription factor. In contrast, DSCR1-1L, regulated by a different promoter than DSCR1-4, activates NFAT and its proangiogenic activity is inhibited by cyclosporin, an inhibitor of calcineurin. In sum, DSCR1-1L, unlike DSCR1-4, potently activates angiogenesis and could be an attractive target for antiangiogenesis therapy.

Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters.

For investigating the expression of cancer/testis (CT) antigens in patients with hepatocellular carcinoma (HCC) in China, and evaluating the correlations between the expression of these CT antigens and clinical parameters, we collected tumors and adjacent non-cancerous tissues of 43 HCC patients from Beijing and 30 HCC patients from Guangxi province. Expression of the mRNA of 14 CT antigens was evaluated by reverse transcription PCR (RT-PCR). The correlation between CT antigen expression and clinical parameters was statistically analyzed. The mRNA expression frequencies of CT antigens in tumor tissue were: MAGE-A1, 69.9%; MAGE-A3, 47.9%; MAGE-A4, 20.0%; MAGE-A10, 36.7%; SSX-1, 67.4%; SSX-2, 35.6%; SSX-4, 48.8%; SSX-5, 30.2%; NY-ESO-1, 42.5%; MAGE-B1, 52.0%; MAGE-B2, 60.0%; MAGE-C1, 48.0%; MAGE-C2, 68.0%; and SCP-1, 33.3%. However, in adjacent tissues, no CT antigen mRNA expression was detected, except SSX-1 in 9.3% patients. In each HCC tissue, the expression of a minimum of one, two, or three CT antigens was in the range of 80-90, 70-80 or 50-70%, respectively. MAGE-A3 mRNA expression differed between the HCC patients in Beijing and Guangxi (P=0.002). The average age of the HCC patients bearing CT antigen positive tumors was higher than that of the HCC patients bearing CT antigen negative tumors. The expression of MAGE-A3, SSX-1, SSX-2, SSX-4, MAGE-B2, MAGE-C1, and MAGE-C2 correlated significantly with older age (P<0.05). Moreover, the expressions of MAGE-A4 and SCP-1 were related to alpha-fetoprotein abnormality (P<0.05), and the expression of NY-ESO-1 was related to early tumor stage (P<0.05). There was no correlation observed between the expression of CT antigens and the sex, HBV infection or tumor size.

A Polymer-Based Antibody-Vinca Drug Conjugate Platform: Characterization and Preclinical Efficacy.

Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development.

Genetic changes and expression of the mannose 6-phosphate/insulin-like growth factor II receptor gene in human hepatitis B virus-associated hepatocellular carcinoma.

It was reported that 60-70% of hepatitis B virus (HBV)-negative hepatocellular carcinoma (HCC) had loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus and this gene was mutated in 55% of these patients with LOH. In this study, genomic DNA from 29 pairs of HBV-positive HCC and corresponding non-tumor tissues was used to analyze LOH at the M6P/IGF2R locus and single deoxyguanosine deletion in this gene by PCR. Total RNA from 19 of the 29 patients was utilized to determine a 192 bp insert in the M6P/IGF2R mRNA and expression of this gene by RT-PCR. Twenty-eight of 29 (97%) HBV-positive HCC were found to be informative at the M6P/IGF2R locus but LOH at this region was only detected in 4/28 (14%) informative patients. Neither single deoxyguanosine deletion in this gene nor 192 bp insert in its mRNA occurred in these patients. Compared with corresponding non-tumor tissues, expression of the M6P/IGF2R mRNA was decreased in 13/19 (68%) HBV-positive HCC tissues, suggesting that M6P/IGF2R may be involved in HBV-associated hepatocarcinogenesis by the regulation of its expression level. In the development of HBV-associated HCC, M6P/IGF2R mutation may not be a major agent.

Alteration of p53 and p21 during hepatocarcinogenesis in tree shrews.

AIM: To investigate p53 mutation and p21 expression in hepatocarcinogenesis induced by hepatitis B virus (HBV) and aflatoxin B(1) (AFB(1)) in tree shrews, and to reveal the role of these genes in hepatocarcinogenesis. METHODS: Tree shrews were divided into four groups: group A, those infected with HBV and fed with AFB(1) (n = 39); group B, those infected with HBV alone (n = 28); group C, those fed with AFB(1) alone (n = 29); and group D, normal controls (n = 20). The tree shrews underwent liver biopsies once every 15 wk. Expression of p53 and p21 proteins and genes in the biopsies and tumor tissues of the experimental tree shrews was detected, respectively, by immunohistochemistry, and by Southern blotting and reverse transcription-polymerase chain reaction and sequencing. RESULTS: The incidence of hepatocellular carcinomas (HCC) was higher in group A (66.7%) than that in group B (3.57%) and C (30%). The time of HCC occurrence was also earlier in group A than that in group C (120.0+/-16.6 wk vs 153.3+/-5.8 wk, respectively, P<0.01). p53 protein was not detected by immunohistochemistry in all groups before the 75(th) wk of the experiment. At the 105(th) wk, the positive rates fo p53 were 78.6%, 60% and 71.4% in groups A, B and C, respectively, which were significantly higher than that in group D (10%) (all P<0.05). An abnormal band of p53 gene was observed in groups A and C. The mutation points of p53 gene in tree shrews with HCC were at codons 275, 78 and 13. The nucleotide sequence and amino acid sequence of tree shrew's wild-type p53 showed 91.7% and 93.4% homologies with those of human p53, respectively. The immunopositivity for p21 was found before HCC development. The incidence of HCC was significantly higher in tree shrews that were positive for p21 than those negative for p21 (80.0% vs 11.0%, P<0.001). The incidence of HCC in p21 positive animals in group A was significantly higher than those positive for p21 in group C (P<0.05). CONCLUSION: A remarkable synergistic effect on HCC development exists between HBV and AFB(1). p53 mutation promotes the development of HCC. HBV and AFB(1) may synergistically induce p53 gene mutation, and stimulate ras gene expression. ras gene is activated at the earlier stage during hepatocarcinogenesis. p21 protein may be an early marker, and the alterations of p53 may be a late event in the development of HCC.

Differential expression of genes during aflatoxin B(1)-induced hepatocarcinogenesis in tree shrews.

AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B(1) (AFB(1)), to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism. METHODS: Tree shrews (Tupaia belangeri chinensis) were treated with or without AFB(1) for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding non-cancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of Atlas(TM) cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared. RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as "important genes" because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC. CONCLUSION: A considerable number of genes could change their expressing levels both in HCC and in HCC-surrounding non-cancerous liver tissues. A few modular genes were up-regulated only in HCC but not in surrounding liver tissues, while some apoptosis-related genes were down-regulated in HCC and up-regulated in surrounding liver tissues. To compare gene-expressing levels among the liver tissues taken at different time points during hepatocarcinogenesis may be helpful to locate the responsible gene (s) and understand the mechanism for AFB(1) induced liver cancer.

Alteration of the p53 gene during tree shrews' hepatocarcinogenesis.

OBJECTIVE: To detect the expression and variation of the p53 gene in hepatocarcinogenesis of tree shrews induced by hepatitis B virus (HBV) and aflatoxin B1 (AFB1). METHODS: Tree shrews were divided into four groups: group A, infected with HBV and fed with AFB1; group B, only infected with HBV; group C, fed with AFB1 alone; and group D normal control. The tree shrews underwent liver biopsy every 15 weeks. Liver and tumor tissues were detected by immunohistochemistry and molecular biotechnologies. RESULTS: The incidence of hepatocellular carcinoma (HCC) was higher in group A (66.7%) than in groups B (0) and C (30%). HCC occurrence was earlier in group A than in group C (120.0+/-16.6 wk vs 153.3+/-5.8 wk, t=3.336, P<0.01). Mutated p53 protein was not found in all groups before 75 weeks of experiment. At the 105th week, the expression rates of mutated p53 protein were 78.6%, 60.0% and 71.4% in groups A, B and C respectively, which were significantly higher than that in group D (10%) (chi2> or =5.03, P<0.05). An abnormal band of the p53 gene was detected in groups A and C. The mutational points of the p53 gene in liver cancer of tree shrews were at codon 275, 78 and 13. Nucleotide sequence and amino acids sequence of tree shrew's wild-type p53 were 91.7% and 93.4% in homology compared with those of human p53, respectively. CONCLUSIONS: Remarkable synergistic effect on HCC exists between HBV and AFB1. Mutated p53 protein expressed before occurrence of HCC promotes the development of HCC. HBV and AFB1 may synergistically induce p53 gene mutation.

[Expression of tumor-specific cancer/testis antigens in hepatocellular carcinoma].

OBJECTIVE: (1) To investigate the expression and gene diversity of the 7 major cancer/testis (CT) antigens, MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1, in hepatocellular carcinoma (HCC). (2) To analyze the correlations between the clinical characters and CT antigens' expression. METHODS: The cancer and para-cancer tissues were collected from 30 HCC patients. The mRNAs of seven CT antigens were detected by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers. The PCR products were sequenced to analyze the CT genes. RESULTS: The MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1 were expressed in 66.7%, 70.0%, 20.0%, 36.7%, 40.0%, 33.3% and 33.3% of the tumor tissues from HCC patients respectively, however, they were not expressed in the para-cancer tissues. Among the 30 patients investigated, 90.0% expressed one CT gene at least, 70.0% expressed two CT genes, and 53.3% expressed three CT genes of the seven CT genes. The coding genes of these CT antigens were highly conserved between in Chinese patients and patients abroad. There were discernible correlations between alpha-fetoprotein level and MAGE-10 or SCP-1 expression level, as well as between average age and MAGE-3 or SSX-2 expression levels (P<0.05). CONCLUSIONS: With a highly conserved coding gene, seven CT antigens were expressed in 20.0% - 70.0% of Chinese HCC patients. CT antigens' expression had correlations with some clinical characters.