抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

https://www.pnas.org/content/pnas/93/18/9821.full.pdf

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands

The discovery of numerous hypermethylated promoters of tumour-suppressor genes, along with a better understanding of gene-silencing mechanisms, has moved DNA methylation from obscurity to recognition as an alternative mechanism of tumour-suppressor inactivation in cancer. Epigenetic events can also facilitate genetic damage, as illustrated by the increased mutagenicity of 5-methylcytosine and the silencing of the MLH1 mismatch repair gene by DNA methylation in colorectal tumours. We review here current mechanistic understanding of the role of DNA methylation in malignant transformation, and suggest Knudson's two-hit hypothesis should now be expanded to include epigenetic mechanisms of gene inactivation.

Cancer-epigenetics comes of age

Platelet-derived growth factor (PDGF)-B-deficient mouse embryos were found to lack microvascular pericytes, which normally form part of the capillary wall, and they developed numerous capillary microaneurysms that ruptured at late gestation. Endothelial cells of the sprouting capillaries in the mutant mice appeared to be unable to attract PDGF-Rbeta-positive pericyte progenitor cells. Pericytes may contribute to the mechanical stability of the capillary wall. Comparisons made between PDGF null mouse phenotypes suggest a general role for PDGFs in the development of myofibroblasts.

https://science.sciencemag.org/content/sci/277/5323/242.full.pdf

Pericyte Loss and Microaneurysm Formation in PDGF-B-Deficient Mice

We are in an era where the potential exists for deriving comprehensive profiles of DNA alterations characterizing each form of human cancer. Such profiles would provide invaluable insight into mechanisms underlying the evolution of each tumor type and will provide molecular markers, which could radically improve cancer detection. To date, no one type of DNA change has been defined which accomplishes this purpose. Herein, by using a candidate gene approach, we show that one category of DNA alteration, aberrant methylation of gene promoter regions, can enormously contribute to the above goals. We have now analyzed a series of promoter hypermethylation changes in 12 genes (p16(INK4a), p15(INK4b), p14(ARF), p73, APC,(5) BRCA1, hMLH1, GSTP1, MGMT, CDH1, TIMP3, and DAPK), each rigorously characterized for association with abnormal gene silencing in cancer, in DNA from over 600 primary tumor samples representing 15 major tumor types. The genes play known important roles in processes encompassing tumor suppression, cell cycle regulation, apoptosis, DNA repair, and metastastic potential. A unique profile of promoter hypermethylation exists for each human cancer in which some gene changes are shared and others are cancer-type specific. The hypermethylation of the genes occurs independently to the extent that a panel of three to four markers defines an abnormality in 70-90% of each cancer type. Our results provide an unusual view of the pervasiveness of DNA alterations, in this case an epigenetic change, in human cancer and a powerful set of markers to outline the disruption of critical pathways in tumorigenesis and for derivation of sensitive molecular detection strategies for virtually every human tumor type.

https://cancerres.aacrjournals.org/content/canres/61/8/3225.full.pdf

A Gene Hypermethylation Profile of Human Cancer

Polyamines in rapid growth and cancer

The polyamines putrescine, spermidine and spermine represent a group of naturally occurring compounds exerting a bewildering number of biological effects, yet despite several decades of intensive research work, their exact physiological function remains obscure. Chemically these compounds are organic aliphatic cations with two (putrescine), three (spermidine) or four (spermine) amino or amino groups that are fully protonated at physiological pH values. Early studies showed that the polyamines are closely connected to the proliferation of animal cells. Their biosynthesis is accomplished by a concerted action of four different enzymes: ornithine decarboxylase, adenosylmethionine decarboxylase, spermidine synthase and spermine synthase. Out of these four enzyme, the two decarboxylases represent unique mammalian enzymes with an extremely short half life and dramatic inducibility in response to growth promoting stimuli. The regulation of ornithine decarboxylase, and to some extent also that of adenosylmethionine decarboxylase, is complex, showing features that do not always fit into the generally accepted rules of molecular biology. The development and introduction of specific inhibitors to the biosynthetic enzymes of the polyamines have revealed that an undisturbed synthesis of the polyamines is a prerequisite for animal cell proliferation to occur. The biosynthesis of the polyamines thus offers a meaningful target for the treatment of certain hyperproliferative diseases, most notably cancer. Although most experimental cancer models responds strikingly to treatment with polyamine antimetabolites--namely, inhibitors of various polyamine synthesizing enzymes--a real breakthrough in the treatment of human cancer has not yet occurred. It is, however, highly likely that the concept is viable. An especially interesting approach is the chemoprevention of cancer with polyamine antimetabolites, a process that appears to work in many experimental animal models. Meanwhile, the inhibition of polyamine accumulation has shown great promise in the treatment of human parasitic diseases, such as African trypanosomiasis.

Polyamines: from Molecular Biology to Clinical Applications

Germline mutations of the putative tumor suppressor gene APC are associated in high frequency with the familial adenomatous polyposis, predisposing the patients to colorectal neoplasia. Similarly, sequence analyses have revealed that in more than half of patients with sporadic colorectal carcinoma or adenoma, the APC gene was mutated. By employing genomic sequencing, i.e., base-specific analysis of methylated cytosines, we show here that the promoter region of the APC gene is heavily methylated at CpG sites in patients with colorectal carcinoma in comparison with normal colonic mucosa and premalignant adenomas. Our results suggest that cytosine methylation of the regulatory sequences of the APC gene could be involved in the progression of human colorectal cancer.

Hypermethylation of the APC (adenomatous polyposis coli) gene promoter region in human colorectal carcinoma

Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1′-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous “priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect.

Juhani Jänne

Papers

Polyamines in rapid growth and cancer

Polyamines: from Molecular Biology to Clinical Applications

Hypermethylation of the APC (adenomatous polyposis coli) gene promoter region in human colorectal carcinoma

Intracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone)

Endothelial-specific gene expression directed by the tie gene promoter in vivo