Cure models as a useful statistical tool for analyzing survival

Cure models are a popular topic within statistical literature but are not as widely known in the clinical literature. Many patients with cancer can be long-term survivors of their disease, and cure models can be a useful tool to analyze and describe cancer survival data. The goal of this article is to review what a cure model is, explain when cure models can be used, and use cure models to describe multiple myeloma survival trends. Multiple myeloma is generally considered an incurable disease, and this article shows that by using cure models, rather than the standard Cox proportional hazards model, we can evaluate whether there is evidence that therapies at the University of Arkansas for Medical Sciences induce a proportion of patients to be long-term survivors.     

Germline translocations in mice: unique tools for analyzing gene function and long-distance regulatory mechanisms

Translocations have provided invaluable tools for identifying both cancer-linked genes and loci associated with heritable human diseases, but heritable human translocations are rare and few mouse models exist. Here we report progress on analysis of a collection of heritable translocations generated by treatment of mice with specific chemicals or radiation during late spermatogenic stages. The translocation mutants exhibit a range of visible phenotypes reflecting the disruption of coding sequences or the separation of genes from essential regulatory elements. The breakpoints of both radiation-induced and chemically induced mutations in these mice are remarkably clean, with very short deletions, duplications, or inversions in some cases, and ligation mediated by microhomology, suggesting nonhomologous end joining as the major path of repair. These mutations provide new tools for the discovery of novel genes and regulatory elements linked to human developmental disorders and new clues to the molecular basis of human genetic disease.     

Immunocytochemical analysis of DNA adducts in single cells: a new tool for experimental carcinogenesis, chemotherapy and molecular epidemiology

The immunocytochemical staining of carcinogen-DNA adducts by a double peroxidase-anti-peroxidase (PAP) method is critically described. It is a powerful new tool for the investigation of the initial processes of chemical carcinogenesis--such as metabolic activation of carcinogens, and modification/repair of DNA--at the level of individual, putative target cell types. It is the method of choice if the cell populations are too small for determination of adducts in isolated DNA, or if information on the tissue distribution of DNA damage is needed. Advantages of the peroxidase staining endpoint over immunofluorescence are its stability on storage of slides and the possibility of evaluation by a conventional microscope. First attempts to quantify staining intensity by microdensitometric equipment are described.     

Role of gene knockout mice in understanding the mechanisms of chemical toxicity and carcinogenesis.

Most chemical carcinogens require metabolic activation to electrophilic metabolites that are capable of binding to DNA and causing gene mutation. Carcinogen metabolism is carried out by large groups of xenobiotic-metabolizing enzymes that include the phase I cytochromes P450 (P450) and phase II enzymes that include various transferases. During the past 10 years, considerable attention has been focused on the role of P450s in human cancer susceptibility. Polymorphisms in expression of P450s and transferases exist in humans and these might render increased susceptibility or resistance to cancer. Thus it is important to understanding how P450s participate in the carcinogenesis process and to determine if they are indeed the rate limiting and critical interface between the chemical and its biological activity. Since there are marked species differences in expressions and catalytic activities of the multiple P450 forms that activate carcinogens, this validation process becomes especially difficult. To address the role of P450s in whole animal carcinogenesis, mice were produced that lack the P450s known to catalyze carcinogen activation. Mouse lines having disruption of genes encoding P450s CYP1A2, CYP2E1, and CYP1B1 were developed by use of gene disruption in empbryonic stem cells. Mice lacking expression of microsomal epoxide hydrolase and NADPH:quinone oxidoreductase were also made. These mice exhibit no grossly abnormal phenotypes, suggesting that the xenobiotic-metabolizing enzymes have no critical roles in mammalian development and physiological homeostasis. This explains the occurrence of polymorphisms in humans and other mammalian species. However, these null mice do show differences in sensitivities to acute chemical toxicities, thus establishing the importance of xenobiotic metabolism in activation pathways that lead to cell death. Rodent bioassays using null mice and known genotoxic carcinogens should establish whether these enzymes are required for carcinogenesis in an intact animal model. These studies will also provide a framework for the production of transgenic mice and carcinogen bioassay protocols that may be more predictive for identifying human carcinogens and validate the molecular epidemiology studies ongoing in humans that seek to establish a role for polymorphisms in cancer risk.     

Reduced mammary gland carcinogenesis in transgenic mice expressing a growth hormone antagonist

Several reports have provided evidence that body size early in life is positively correlated with risk of subsequent breast cancer, but the biological basis for this relationship is unclear. We examined tumour incidence in transgenic mice expressing a growth hormone (GH) antagonist and in non-transgenic littermates following exposure to dimethylbenz[ a ]anthracene (DMBA), a well characterized murine mammary gland carcinogen. The transgenic animals had lower IGF-I levels, were smaller in terms of body size and weight, and exhibited decreased tumour incidence relative to controls. The demonstration that both body size early in life and breast cancer incidence are influenced by experimental perturbation of the GH-IGF-I axis in a transgenic model provides evidence that variability between individuals with respect to these hormones underlies the relationship between body size early in life and breast cancer risk observed in epidemiological studies.     

绿色荧光蛋白转基因小鼠用作hprt基因突变试验动物模型的研究

目的： 探讨绿色荧光蛋白 (green fluorescent protein,GFP)转基因小鼠用作hprt基因突变试验动物模型的可能性。方法：用乙基亚硝基脲 (N-ethyl-N-nitrosourea,ENU)诱导GFP转基因小鼠和普通小鼠基因突变,比较诱导后两种小鼠的嗜多染红细胞微核率变化情况。再观察GFP小鼠细胞克隆对GFP转基因小鼠hprt基因的DNA和mRNA序列进行验证,并对GFP转基因小鼠hprt突变克隆外显子1和外显子9进行了双向测序。结果：ENU诱导后GFP转基因小鼠和普通小鼠的微核率变化趋势相同。GFP转基因小鼠的微孔培养物比普通小鼠的微孔培养物易于确认,且降低了假阳性率和假阴性率。GFP转基因小鼠的hprt基因是完整的,与普通小鼠相比未见明显差异。GFP转基因小鼠hprt基因6个突变克隆外显子1的序列均与野生型小鼠细胞的序列一致,3个突变克隆外显子9的序列中有2个发生了A∶T→T∶A颠换。结论：ENU诱导的GFP转基因小鼠突变模型良好,GFP转基因小鼠可用作小鼠淋巴细胞hprt基因突变试验的动物模型,准确性高且试验难度低。     

Short interfering RNAs as a tool for cancer gene therapy

There are mainly two types of short RNAs that target complementary messengers in animals: small interfering RNAs and micro-RNAs. Both are produced by the cleavage of double-stranded RNA precursors by Dicer, a member of the Rnase III family of double-stranded specific endonucleases, and both guide the RNA-induced silencing complex to cleave specifically RNAs sharing sequence identity with them. In designing a particular RNA interference (RNAi), it is important to identify the sense/antisense combination that provides the most potent suppression of the target mRNA, and several rules have been established to give >90% gene expression inhibition. RNAi technology can be directed against cancer using a variety of strategies. These include the inhibition of overexpressed oncogenes, blocking cell division by interfering with cyclin E and related genes or promoting apoptosis by suppressing antiapoptotic genes. RNAi against multidrug resistance genes or chemoresistance targets may also provide useful cancer treatments. Studies investigating these approaches in preclinical models are also reviewed.     

Chemically induced skin carcinogenesis in a transgenic mouse line (TGAC) carrying a v-Ha-ras gene

A transgenic mouse line (TGAC) created in the FVB/N strain,carries a v-Ha-ras gene fused to     

Helicobacter pylori and molecular mechanisms of gastric carcinogenesis: targets for prevention and therapy

Gastric cancer is still a leading cause of cancer death worldwide. It is frequently diagnosed in an advanced stage and the overall prognosis is dismal. A major improvement will only be reached with a better understanding of the genetic changes underlying gastric cancer, which may provide us with effective prevention and treatment strategies. This review will highlight the present knowledge on the role of Helicobacter pylori in gastric carcinogenesis and the molecular pathogenesis of gastric cancer, as well as provide a brief overview of possible future targets for more specific and successful treatment options.     

Immunosurveillance of Erbb2 carcinogenesis in transgenic mice is concealed by a dominant regulatory T-cell self-tolerance

To assess the role of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells in overcoming immunosurveillance of Erbb2 (HER-2/neu) mammary lesions, we studied the effects of their sustained removal in BALB/c female mice made transgenic for the rat Erbb2 (r-Erbb2) oncogene (BALB-neuT mice), which develop multiple mammary carcinomas. During the progression of these lesions, Treg cells expand in the spleen, tumor draining lymph nodes, and tumors. Repeated administration of anti-CD25 antibodies extends tumor-free survival, reduces carcinoma multiplicity, and leads to the manifestation of a natural antibody and CTL-mediated reactivity against r-Erbb2. Loss of Foxp3(+) Treg cells during anti-CD25 treatment remarkably caused the disappearance of Gr1(+) immature myeloid cells, suggesting a cross-talk between these two inhibitory immune cell types. Treg cell expansion associated with r-Erbb2 overexpression may be seen as a physiologic response to dampen the immune reaction elicited by local anomalous overexpression of a self-antigen.     

Tissue-specific carcinogenesis in transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation

Germ line mutations of the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2A (MEN 2A), an inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. To study the mechanism of tissue-specific tumor development by RET with a MEN2A (cysteine 634-->arginine) mutation, we generated transgenic mice by introducing the RET-MEN2A gene fused to Moloney murine leukemia virus long terminal repeat. Expression of the transgene and its product was detected at variable levels in a variety of tissues including thyroid, heart, liver, colon, parotid gland, and brain. All of 29 mice analyzed developed thyroid C-cell hyperplasia or medullary carcinoma, accompanying high levels of serum calcitonin. In addition, development of mammary or parotid gland adenocarcinoma was observed in one-half of the transgenic mice. RET dimerization and its complex formation with Shc and Grb2 adaptor proteins were detected in tumor tissues. Unexpectedly, no tumor formation was found in other tissues despite RET-MEN2A expression where RET dimerization was undetectable. Because these tissues but not tumors expressed glial cell line-derived neurotrophic factor family receptor alpha (GFR alpha) at high levels, this suggested that GFR alpha expression may interfere in the dimerization of the RET-MEN2A mutant proteins, leading to tissue-specific tumor development in vivo.     

Androgen-dependent mammary carcinogenesis in rats transgenic for the Neu proto-oncogene

Transgenic rats were created with overexpression of the Neu proto-oncogene in the mammary gland of both sexes, yet only males developed mammary cancer in an androgen-dependent fashion. Transgenic females only developed mammary cancer if treated with androgens. These tumors were positive for androgen receptor (AR), but negative for estrogen and progesterone receptors. Extensive analysis failed to detect mutations anywhere within the neu transgene from mammary carcinomas. Established mammary carcinomas eventually escaped their dependency on androgens. Transgenic long-term gonadectomized rats did not develop mammary cancer, but Neu overexpression stimulated the growth of their mammary glands. Our results suggest crosstalk between the Neu proto-oncogene and AR signaling pathways in the growth of both the normal and cancerous mammary epithelium.     

Diet-dependent carcinogenesis of pancreatic islets and liver in transgenic mice expressing oncogenes under the control of the L-type pyruvate kinase gene promoter.

The liver-type pyruvate kinase (L-PK) gene is controlled positively by insulin and carbohydrates, negatively by glucagon and fasting. Diet-inducible models of carcinogenesis were obtained using the L-PK gene promoter and regulatory sequences to control the expression of c-myc and SV40 T oncogenes in transgenic mice. L-PK/c-myc and L-PK/Tag animals fed a carbohydrate-rich diet developed hepatocarcinomas. In addition, L-PK/Tag animals developed diet-dependent, aggressive endocrine pancreatic tumors, preceded by islet hyperplasia involving the different analysed cell populations (alpha, beta and delta). Expression of the L-PK gene was demonstrated in pancreatic tumors, in rat isolated islets and in rat insulinoma-derived cells (RIN line), revealing a new tissue specificity of the L-PK gene. Our results suggest that this gene may be expressed in islet progenitor cells from which the different mature endocrine cells derive.     

Cytosine deaminase gene as a potential tool for the genetic therapy of colorectal cancer

The bacterial enzyme cytosine deaminase (CD) catalyzes the conversion of 5-fluorocytosine (5-FC) to the lethal 5-fluorouracil (5-FU) and so provides a useful system for selective killing of gene-modified mammalian tumor cells. Cloning of the CD gene from Escherichia coli and expression in human tumor cell lines enabled these cells to convert ³H-labeled 5-FC into ³H-5-FU. Two CD-expressing human tumor cell lines (adenocarcinoma cell line KM12 and glioblastoma cell line T1115) became 200-fold more sensitive to 5-FC than the nonexpressing parental cell lines. At least 90% of the cells are killed within 7 days. CD-expressing cells are able to kill nonexpressing cells when grown in the same culture flask (bystander effect). The CD gene may be used as a suicide system for in situ chemotherapy or as a safety mechanism abrogating the expression of other genes. © 1996 Wiley-Liss, Inc.     

Mammary carcinogenesis in transgenic mice expressing a dominant-negative mutant of DNA polymerase beta in their mammary glands

DNA polymerase beta (polbeta) is a major contributor to mammalian DNA damage repair through its gap-filling DNA synthesis and 5'-deoxyribose phosphate lyase activities. In this way, polbeta plays pivotal roles in the repair of oxidative DNA damage, replication, embryonic survival, neuronal development, meiosis, apoptosis and telomere function. A 36 kDa truncated polbetaDelta protein is expressed in human colorectal, breast, lung and renal carcinomas, but not in normal matched tissues. Interestingly, a binary protein-protein complex of polbetaDelta and X-ray cross-complementing group 1 acts as dominant-negative mutant. In this study, the potential tumorigenic activity of polbetaDelta was examined in nude and transgenic mouse models. Mouse embryonic fibroblasts (MEFs) expressing polbetaDelta in the absence of endogenous polbeta exhibited increased susceptibility to N-methyl-N-nitrosourea (MNU)-induced morphological transformation as compared with cells expressing wild-type (WT) polbeta. This was accompanied by reduced gap-filling DNA synthesis activity. Anchorage-independent transformed cells derived from polbetaDelta-expressing MEFs induced 100% tumor occurrence in nude mice. To support these data, we established transgenic mice expressing polbetaDelta specifically in the mammary glands from a whey acidic protein promoter-driven transgene. This is the first report of transgenic mice with tissue-specific expression of polbetaDelta. MNU-induced tumor formation was analyzed in transgenic mice expressing polbetaDelta together with endogenous WT polbeta in their mammary glands and in normal control mice expressing only WT polbeta. The latent period of tumor appearance was markedly shorter and tumor incidence was significantly higher in transgenic animals than in control animals treated under the same conditions. These results indicate that cells expressing the mutant polbetaDelta display an enhanced sensitivity to MNU that probably underlies an increased susceptibility to tumorigenesis.     

Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin

Background:

Although pemetrexed, a potent thymidylate synthase (TS) inhibitor, enhances the cytoytoxic effect of platinum compounds against malignant pleural mesothelioma (MPM), novel combinations with effective targeted therapies are warranted. To this end, the current study evaluates new targeted agents and their pharmacological interaction with carboplatin–pemetrexed in human MPM cell lines.

Methods:

We treated H2052, H2452, H28 and MSTO-211H cells with carboplatin, pemetrexed and targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, enzastaurin and ZM447439) and evaluated the modulation of pivotal pathways in drug activity and cancer cell proliferation.

Results:

Vandetanib emerged as the compound with the most potent cytotoxic activity, which interacted synergistically with carboplatin and pemetrexed. Drug combinations blocked Akt phosphorylation and increased apoptosis. Vandetanib significantly downregulated epidermal growth factor receptor (EGFR)/Erk/Akt phosphorylation as well as E2F-1 mRNA and TS mRNA/protein levels. Moreover, pemetrexed decreased Akt phosphorylation and expression of DNA repair genes. Finally, most MPM samples displayed detectable levels of EGFR and TS, the variability of which could be used for patients'' stratification in future trials with vandetanib–pemetrexed–carboplatin combination.

Conclusion:

Vandetanib markedly enhances pemetrexed–carboplatin activity against human MPM cells. Induction of apoptosis, modulation of EGFR/Akt/Erk phosphorylation and expression of key determinants for pemetrexed and carboplatin activity contribute to this synergistic interaction, and, together with the expression of these determinants in MPM samples, warrant further clinical investigation.