Biomedical subjects
Christoph Lengauer
Publications and source records attributed to Christoph Lengauer.
The Chk2 tumor suppressor is not required for p53 responses in human cancer cells.
Ionizing radiation damages chromosomal DNA and activates p53-dependent transcription in mammalian cells. The Chk2 protein kinase has been hypothesized to be the primary mediator of this response. We have rigorously tested this hypothesis in human cells by disrupting the CHK2 gene through homologous recombination. We found that the p53 response was unexpectedly robust in such cells. Phosphorylation of p53 at serine 20, accumulation of p53 protein, transcriptional activation of p53 target genes, and cell cycle arrest and apoptotic death phenotypes were completely intact regardless of CHK2 status. Our results indicate that Chk2 kinase is not required for p53 activation in human cells and explain why CHK2 and TP53 mutations can jointly occur in human tumors.
The significance of unstable chromosomes in colorectal cancer.
A very large fraction of cancers have an abnormal genetic content, called aneuploidy, which is characterized by changes in chromosome structure and number. One explanation for this aneuploidy is chromosomal instability, in which cancer cells gain or lose whole chromosomes or large fractions of chromosomes at a greatly increased rate compared with normal cells. Here, we explore experimental and theoretical evidence for the initiation of chromosomal instability in very early colorectal cancers, and reflect on the role that chromosomal instability could have in colorectal tumorigenesis.
Digital karyotyping.
Alterations in the genetic content of a cell are the underlying cause of many human diseases, including cancers. We have developed a method, called digital karyotyping, that provides quantitative analysis of DNA copy number at high resolution. This approach involves the isolation and enumeration of short sequence tags from specific genomic loci. Analysis of human cancer cells by using this method identified gross chromosomal changes as well as amplifications and deletions, including regions not previously known to be altered. Foreign DNA sequences not present in the normal human genome could also be readily identified. Digital karyotyping provides a broadly applicable means for systematic detection of DNA copy number changes on a genomic scale.
The role of chromosomal instability in tumor initiation.
Chromosomal instability (CIN) is a defining characteristic of most human cancers. Mutation of CIN genes increases the probability that whole chromosomes or large fractions of chromosomes are gained or lost during cell division. The consequence of CIN is an imbalance in the number of chromosomes per cell (aneuploidy) and an enhanced rate of loss of heterozygosity. A major question of cancer genetics is to what extent CIN, or any genetic instability, is an early event and consequently a driving force for tumor progression. In this article, we develop a mathematical framework for studying the effect of CIN on the somatic evolution of cancer. Specifically, we calculate the conditions for CIN to initiate the process of colorectal tumorigenesis before the inactivation of tumor suppressor genes.
Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status.
Genes of the RAF family encode kinases that are regulated by Ras and mediate cellular responses to growth signals. Activating mutations in one RAF gene, BRAF, have been found in a high proportion of melanomas and in a small fraction of other cancers. Here we show that BRAF mutations in colorectal cancers occur only in tumours that do not carry mutations in a RAS gene known as KRAS, and that BRAF mutation is linked to the proficiency of these tumours in repairing mismatched bases in DNA. Our results not only provide genetic support for the idea that mutations in BRAF and KRAS exert equivalent effects in tumorigenesis, but also emphasize the role of repair processes in establishing the mutation spectra that underpin human cancer.
DNMT1 and DNMT3b cooperate to silence genes in human cancer cells.
Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.
Targeted inactivation of p53 in human cells does not result in aneuploidy.
Because p53 mutation and aneuploidy usually coexist, it has been suggested that p53 inactivation leads to aneuploidy. We have rigorously tested this hypothesis in diploid human cell lines in which p53 was experimentally inactivated by targeted homologous recombination. Cells completely deficient in p53 did not become aneuploid, although a slight tendency toward tetraploidization was observed. No increased rates of numerical or structural chromosomal instabilities were observed in the p53-deficient cells. Rates of sister chromatid exchange and homologous recombination were also unaffected by p53 status. These results show that inactivation of p53 does not, in and of itself, lead to the development of aneuploidy.
Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies.
A classification of neoplasms as chromosomally stable vs. unstable is of importance in assessing inherited risks but is primarily defined in cell lines where rates of instability can be directly measured. The confirmation of such a mechanistic theory in primary tissues is desirable. We examined anaphase figures in histologic slides to evaluate such criteria. The process of chromosomal instability (CIN) involves mis-segregation of chromosomes at anaphase in some or most instances, whereas chromosomally stable tumors should lack these particular abnormalities. Anaphase bridges are associated with chromosome mis-segregation and have occasionally been observed in colorectal neoplasia and in sarcomas, but have not been considered for use as a diagnostic tool. We assembled series of sarcomas, colorectal and pancreatic carcinomas belonging to two groups: those associated with or lacking chromosomal instability. Among the chromosomally stable tumors, all lacked anaphase bridges, while anaphase bridges were found in most sarcomas and carcinomas having complex genomic alterations. These results confirm a mechanistic explanation involving chromosome mis-segregation for primary human cancers having aneuploidy/CIN. A basic mechanistic distinction between neoplasms with CIN and those with minimal chromosomal changes is supported. Most importantly, our data suggest a diagnostic utility for the examination of anaphase bridges in the routine practice of pathology with a high potential to impact clinical decision-making processes regarding colorectal cancer treatment.
Dynamics of genetic instability in sporadic and familial colorectal cancer.
Genetic instability is a defining feature of human cancer. In colorectal cancer, two specific types of genetic instabilities have been identified: microsatellite instability (MIN) leads to a 1000-fold increase in the rate of subtle DNA changes, whereas chromosomal instability (CIN) enhances the rate at which gross chromosomal changes occur during cell division. In this paper, we develop a mathematical model for the dynamics of colon cancer initiation. We outline the processes and rate constants that determine the fraction of colon cancers where MIN or CIN mutations precede the inactivation of the first tumor suppressor gene. For a wide range of parameter values, we find support for the radical hypothesis that genetic instability initiates colonic tumorigenesis. We compare sporadic and hereditary forms of colorectal cancer.
Exploring and exploiting instability.
Genetic instability was postulated to be essential for tumor development almost three decades ago, and yet its exact nature in and relationship with cancer continue to be ill-understood and hotly debated subjects. In this article, we review and discuss current knowledge and thinking about the existence, characteristics, reasons for and mechanisms of genetic instability in human cancers, as well as how its study can help us better understand and fight cancer. Particular emphasis is given to chromosomal instability, the most common and least understood of the types of genetic instability found in human tumors.