Guselkumab is superior to fumaric acid esters in patients with moderate-to-severe plaque psoriasis who are naive to systemic treatment: results from a randomized, active-comparator-controlled phase IIIb trial (POLARIS).

BACKGROUND: Guselkumab, a fully human interleukin-23 antibody, is approved for systemic treatment of patients with moderate-to-severe plaque psoriasis. OBJECTIVES: To compare the efficacy and safety of guselkumab with those of fumaric acid esters (FAE) in patients with moderate-to-severe plaque psoriasis who are naive to systemic treatment. METHODS: Eligible patients were randomized to this multicentre, randomized, open-label, assessor-blinded, active-comparator-controlled phase IIIb study to receive guselkumab 100 mg by subcutaneous injection or oral FAE according to local label guidelines. RESULTS: Through week 24, 56 of 60 patients completed guselkumab treatment and 36 of 59 completed FAE treatment. The primary endpoint (proportion of patients with ≥ 90% improvement from their baseline Psoriasis Area and Severity Index; PASI 90 response) was achieved by significantly more patients receiving guselkumab than FAE at week 24 (82% vs. 14%, P < 0·001). Analysis of the major secondary endpoints confirmed a statistically significant difference between the treatments with regards to PASI 75 response (90% vs. 27%, P < 0·001) and Dermatology Life Quality Index score of 0 or 1 (no effect at all on the patient's quality of life; 62% vs. 17%, P < 0·001). More patients in the guselkumab group achieved completely clear skin (PASI 100 response) than in the FAE group (32% vs. 3%, P < 0·001). The incidence of adverse events was lower with guselkumab than with FAE (73% vs. 98%). Overall, 28% of patients on FAE discontinued due to an adverse event, compared with none receiving guselkumab. No new safety findings were observed for guselkumab. CONCLUSIONS: Guselkumab demonstrated superiority over FAE in systemic-treatment-naive patients with moderate-to-severe plaque psoriasis through 24 weeks.

Multitarget Stool DNA Test Performance in an Average-Risk Colorectal Cancer Screening Population.

INTRODUCTION: We set out to evaluate the performance of a multitarget stool DNA (MT-sDNA) in an average-risk colonoscopy-controlled colorectal cancer (CRC) screening population. MT-sDNA stool test results were evaluated against fecal immunochemical test (FIT) results for the detection of different lesions, including molecularly defined high-risk adenomas and several other tumor characteristics. METHODS: Whole stool samples (n = 1,047) were prospectively collected and subjected to an MT-sDNA test, which tests for KRAS mutations, NDRG4 and BMP3 promoter methylation, and hemoglobin. Results for detecting CRC (n = 7), advanced precancerous lesions (advanced adenoma [AA] and advanced serrated polyps; n = 119), and non-AAs (n = 191) were compared with those of FIT alone (thresholds of 50, 75, and 100 hemoglobin/mL). AAs with high risk of progression were defined by the presence of specific DNA copy number events as measured by low-pass whole genome sequencing. RESULTS: The MT-sDNA test was more sensitive than FIT alone in detecting advanced precancerous lesions (46% (55/119) vs 27% (32/119), respectively, P < 0.001). Specificities among individuals with nonadvanced or negative findings (controls) were 89% (791/888) and 93% (828/888) for MT-sDNA and FIT testing, respectively. A positive MT-sDNA test was associated with multiple lesions (P = 0.005), larger lesions (P = 0.03), and lesions with tubulovillous architecture (P = 0.04). The sensitivity of the MT-sDNA test or FIT in detecting individuals with high-risk AAs (n = 19) from individuals with low-risk AAs (n = 52) was not significantly different. DISCUSSION: In an average-risk screening population, the MT-sDNA test has an increased sensitivity for detecting advanced precancerous lesions compared with FIT alone. AAs with a high risk of progression were not detected with significantly higher sensitivity by MT-sDNA or FIT.

Social position and geriatric syndromes among Swedish older people: a population-based study.

BACKGROUND: Older people with a low social position are at higher risk of poor health outcomes compared to those with a higher social position. Whether lower social position also increases the risk of geriatric syndromes (GSs) remains to be determined. This study investigates the association of social position with GSs among older community-dwellers. METHODS: Three consecutive population-based health surveys in 2006, 2010 and 2014 among older community-dwellers (age 65-84 years) in Stockholm County were combined (n = 17,612) and linked with Swedish administrative registry information. Social position was assessed using registry information (i.e. education, country of origin and civil status) and by self-reports (i.e. type of housing and financial stress). GSs were assessed by self-reports of the following conditions: insomnia, urinary incontinence, functional decline, falls, depressive disorder, hearing or vision problems. Binomial logistic regression analyses were used to estimate the association between social position and GSs after adjusting for age, sex, health status, health behavior and social stress. RESULTS: The prevalence of GSs was 70.0%, but varied across GSs and ranged from 1.9% for depression to 39.1% for insomnia. Living in rented accommodation, being born outside the Nordic countries, being widowed or divorced were associated with GS presence. Financial stress was most strongly associated with GSs (adjusted odds ratio, 2.59; 95% CI, 2.13-3.15). CONCLUSION: GSs are highly prevalent among older Swedish community-dwellers with wide variations across syndromes and strong association with all measures of social position, most strikingly that of experiencing financial stress.

Molecular profiling of longitudinally observed small colorectal polyps: A cohort study.

BACKGROUND: Knowledge of the natural history of colorectal adenomas is limited because these lesions are removed upon detection. The few studies in which small adenomas have been left in situ for a limited period of time, have shown that most lesions remain stable or even completely regress. Specific DNA copy number changes ('cancer associated events' or CAEs) are associated with progression of adenomas to cancer. In this study we evaluated whether molecular features of progression correlated with growth of small polyps. METHODS: Small (6-9 mm) colorectal precursor lesions detected on CT-colonography (CTC) were left in situ and re-evaluated with CTC after three years. Based on volumetric change, polyps were classified as either grown, stable or regressed. Surveillance CTC was followed by colonoscopy, during which all lesions were resected. Using DNA isolated from FFPE polyp tissues, low-coverage whole genome sequencing was performed to determine DNA copy number profiles, as well as target enrichment mutation analysis and CpG island methylation phenotype (CIMP) analysis. Expression of DNA mismatch repair (MMR) proteins was determined by immunohistochemistry. Samples were marked as MMR proficient if all MMR proteins were expressed. FINDINGS: Out of 68 polyps resected at colonoscopy, for 65 (96%) material was available. Of these, 31 (48%) had grown, 27 (41%) remained stable and 7 (11%) regressed. Polyps with at least one CAE had higher growth rates compared to polyps without CAEs (difference 91% growth (95% CI 13-169), p = .023). CAEs were absent in lesions that had partially regressed. Mutations occurred in 94% of the polyps, with higher growth rates being associated with polyps having ≥2 mutations compared to lesions with only 0-1 mutations (difference 99% growth (95% CI 9-189), p = .032). All samples were MMR proficient. No relation between growth and CIMP was observed. INTERPRETATION: Molecular alterations associated with colorectal cancer, correlated with growth of small polyps and were absent in polyps that regressed. Therefore, this longitudinal study provides in vivo support in the human setting for the functional role of these molecular alterations, that have mostly been identified by cross sectional observations in tissue samples of colorectal adenomas and cancers. FUND: Alpe d'Huzes- Dutch Cancer Society (project number NKI2013-6338).

Injurious falls and subsequent adverse drug events among elderly - a Swedish population-based matched case-control study.

BACKGROUND: Fall injuries are stressful and painful and they have a range of serious consequences for older people. While there is some clinical evidence of unintentional poisoning by medication following a severe fall injuries, population-based studies on that association are lacking. This is investigated in the current study, in which attention is also paid to different clinical conditions of the injured patients. METHODS: We conducted a matched case-control study of Swedish residents 60 years and older from various Swedish population-based registers. Cases defined as adverse drug events (ADE) by unintentional poisoning leading to hospitalization or death were extracted from the National Patient Register (NPR) and the Cause of Death Register from January 2006 to December 2009 (n = 4418). To each case, four controls were matched by sex, age and residential area. Information on injurious falls leading to hospitalization six months prior to the date of hospital admission or death from ADE by unintentional poisoning, and corresponding date for the controls, was extracted from the NPR. Data on clinical conditions, such as dispensed medications, comorbidity and previous fall injuries were also extracted from the Swedish Prescribed Drug Register (SPDR) and NPR. Effect estimates were calculated using conditional logistic regression and presented as odds ratios (OR) and 95% confidence intervals (CI). RESULTS: We found a three-fold increased risk of unintentional poisoning by medication in the six-month period after an injurious fall (OR 3.03; 95% CI, 2.54-3.74), with the most pronounced increase 1-3 weeks immediately after (OR, 7.66; 95% CI, 4.86-12.1). In that time window, from among those hospitalized for a fall (n = 92), those who sustained an unintentional poisoning (n = 60) tended to be in poorer health condition and receive more prescribed medications than those who did not, although this was not statistically significant. Age stratified analyses revealed a higher risk of poisoning among the younger (aged 60-79 years) than older elderly (80+ years). CONCLUSION: Medication-related poisoning leading to hospitalization or death can be an ADE subsequent to an episode of hospitalization for a fall-related injury. Poisoning is more likely to occur closer to the injurious event and among the younger elderly. It cannot be ruled out that some of those falls are themselves ADE and early signs of greater vulnerability among certain patients.

CLUSEAN: a computer-based framework for the automated analysis of bacterial secondary metabolite biosynthetic gene clusters.

Bacterial secondary metabolites are an important source of antimicrobial and cytostatic drugs. These molecules are often synthesized in a stepwise fashion by multimodular megaenzymes that are encoded in clusters of genes encoding enzymes for precursor supply and modification. In this work,we present an open source software pipeline, CLUSEAN (CLUster SEquence ANalyzer) that helps to annotate and analyze such gene clusters. CLUSEAN integrates standard analysis tools, like BLAST and HMMer, with specific tools for the identification of the functional domains and motifs in nonribosomal peptide synthetases (NRPS)/type I polyketide synthases (PKS) and the prediction of specificities of NRPS.

Galactomannan does not precede major signs on a pulmonary computerized tomographic scan suggestive of invasive aspergillosis in patients with hematological malignancies.

BACKGROUND: Detection of serum galactomannan (GM) antigen and presence of the halo sign on a pulmonary computerized tomographic (CT) scan have a high specificity but a low sensitivity to diagnose invasive aspergillosis (IA) in patients at risk for this disease. To our knowledge, the relationship between the time at which pulmonary infiltrates are detected by CT and the time at which GM antigens are detected by enzyme immunoassay (EIA) has not been studied. METHODS: In a prospective study, tests for detection of GM were performed twice weekly for patients with hematological malignancies who had undergone hematopoetic stem cell transplantation (HSCT) or had received induction and/or consolidation chemotherapy. A pulmonary CT scan was performed once weekly. Infiltrates were defined as either major or minor signs. IA was classified as proven, probable, or possible, in accordance with the definition stated by the European Organization for Research and Treatment of Cancer-Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group. RESULTS: We analyzed 161 episodes of infection in 107 patients (65 allogeneic HSCT recipients, 30 autologous HSCT recipients, and 66 induction and/or consolidation chemotherapy recipients). A total of 109 episodes with no IA, 32 episodes with possible IA, and 20 episodes with probable or proven IA were identified. Minor pulmonary signs were detected by CT in 70 episodes (43%), and major pulmonary signs were detected by CT in 11 episodes (7%). Univariate and multivariate analyses revealed no significant association between detection of GM by EIA and detection of abnormal pulmonary signs by CT. A significant association was found between GM levels and receipt of piperacillin-tazobactam. GM test results were not positive before major signs were seen on CT images. Only 7 (10%) of 70 patients with minor pulmonary signs had positive GM test results before detection of the greatest pathologic change by CT. CONCLUSIONS: We show that detection of GM by EIA does not precede detection of major lesions by pulmonary CT. In the clinical setting, the decision to administer mold-active treatment should based on detection of new pulmonary infiltrates on CT performed early during infection, rather than on results of EIA for detection of GM.

The wheat D-genome HMW-glutenin locus: BAC sequencing, gene distribution, and retrotransposon clusters.

A bacterial-artificial-chromosome (BAC) clone from the genome of Triticum tauschii, the D-genome ancestor of hexaploid bread wheat, was sequenced and the presence of the two paralogous x- and y-type high-molecular-weight (HMW) glutenin genes of the Glu-D1 locus was confirmed. These two genes occur in the same orientation, are 51,893 bp apart, and the separating DNA includes a 31,000-bp cluster of retrotransposons. A second retrotransposon cluster of 32,000 bp follows the x-type HMW-glutenin gene region. Each HMW-glutenin gene is found within a region of mainly unique DNA sequence which includes multiple additional genes including an active endosperm globulin gene not previously reported in the Triticeae family, a leucine-rich-repeat (LRR) type gene truncated at the 5' end of the BAC, a kinase gene of unknown activity, remnants of a paralogous second globulin gene, and genes similar to two hypothetical rice genes. The newly identified globulin genes are assigned to a locus designated Glo-2. Comparison to available orthologous regions of the wheat A and B genomes show rapid sequence divergences flanking the HMW-glutenin genes, and the absence of two hypothetical and unknown genes found 5' to the B-genome x-type ortholog. The region surrounding the Glu-D1 locus is similar to other reported Triticeae BAC sequences; i.e. small gene islands separated by retrotransposon clusters.

A phosphate transporter expressed in arbuscule-containing cells in potato.

Arbuscular mycorrhizas are the most common non-pathogenic symbioses in the roots of plants. It is generally assumed that this symbiosis facilitated the colonization of land by plants. In arbuscular mycorrhizas, fungal hyphae often extend between the root cells and tuft-like branched structures (arbuscules) form within the cell lumina that act as the functional interface for nutrient exchange. In the mutualistic arbuscular-mycorrhizal symbiosis the host plant derives mainly phosphorus from the fungus, which in turn benefits from plant-based glucose. The molecular basis of the establishment and functioning of the arbuscular-mycorrhizal symbiosis is largely not understood. Here we identify the phosphate transporter gene StPT3 in potato (Solanum tuberosum). Functionality of the encoded protein was confirmed by yeast complementation. RNA localization and reporter gene expression indicated expression of StPT3 in root sectors where mycorrhizal structures are formed. A sequence motif in the StPT3 promoter is similar to transposon-like elements, suggesting that the mutualistic symbiosis evolved by genetic rearrangements in the StPT3 promoter.

Software scripts for quality checking of high-throughput nucleic acid sequencers.

We have developed a graphical interface to allow the researcher to view and assess the quality of sequencing results using a series of program scripts developed to process data generated by automated sequencers. The scripts are written in Perl programming language and are executable under the cgibin directory of a Web server environment. The scripts direct nucleic acid sequencing trace file data output from automated sequencers to be analyzed by the phred molecular biology program and are displayed as graphical hypertext mark-up language (HTML) pages. The scripts are mainly designed to handle 96-well microtiter dish samples, but the scripts are also able to read data from 384-well microtiter dishes 96 samples at a time. The scripts may be customized for different laboratory environments and computer configurations. Web links to the sources and discussion page are provided.

Simian virus 40 large tumor antigen modulates the Raf signaling pathway.

The large tumor antigen of simian virus 40 (SVLT) is a potent oncogene. Although inactivation of the p53 and pRb tumor suppressors has been causally linked to the transforming properties of SVLT, its exact mechanism of action remains undefined. Previous data indicated that Ras is activated in SVLT-expressing cells. In this report we show that SVLT also increases Raf kinase activity in both insect and mammalian cells, thus identifying the Raf kinase as an additional target of SVLT. Our results further show that SVLT was still able to activate Raf in cells where Ras levels had been drastically reduced through expression of an antisense construct, indicating that SVLT may activate Raf at least partly by a mechanism that is independent of its stimulatory effect on Ras.

Aneuploidy precedes and segregates with chemical carcinogenesis.

A century ago, Boveri proposed that cancer is caused by aneuploidy, an abnormal balance of chromosomes, because aneuploidy correlates with cancer and because experimental aneuploidy generates "pathological" phenotypes. Half a century later, when cancers were found to be nonclonal for aneuploidy, but clonal for somatic gene mutations, this hypothesis was abandoned. As a result, aneuploidy is now generally viewed as a consequence, and mutated genes as a cause of cancer. However, we have recently proposed a two-stage mechanism of carcinogenesis that resolves the discrepancy between clonal mutation and nonclonal karyotypes. The proposal is as follows: in stage 1, a carcinogen "initiates" carcinogenesis by generating a preneoplastic aneuploidy; in stage 2, aneuploidy causes asymmetric mitosis because it biases balance-sensitive spindle and chromosomal proteins and alters centrosomes both numerically and structurally (in proportion to the degree of aneuploidy). Therefore, the karyotype of an initiated cell evolves autocatalytically, generating ever-new chromosome combinations, including neoplastic ones. Accordingly, the heterogeneous karyotypes of "clonal" cancers are an inevitable consequence of the karyotypic instability of aneuploid cells. The notorious long latent periods, of months to decades, from carcinogen to carcinogenesis, would reflect the low probability of evolving by chance karyotypes that compete favorably with normal cells, in principle analagous to natural evolution. Here, we have confirmed experimentally five predictions of the aneuploidy hypothesis: (1) the carcinogens dimethylbenzanthracene and cytosine arabinoside induced aneuploidy in a fraction of treated Chinese hamster embryo cells; (2) aneuploidy preceded malignant transformation; (3) transformation of carcinogen-treated cells occurred only months after carcinogen treatment, i.e., autocatalytically; (4) preneoplastic aneuploidy segregated with malignant transformation in vitro and with 14 of 14 tumors in animals; and (5) karyotypes of tumors were heterogeneous. We conclude that, with the carcinogens studied, aneuploidy precedes cancer and is necessary for carcinogenesis.

Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis.

An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9. Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K(m) for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function.

Development of a community-based diabetes management program for Pacific Islanders.

PURPOSE: This study examined the perception of diabetes among a sample of Pacific Islanders in Honolulu, Hawaii. All 23 participants were diagnosed with type 2 diabetes, ranged in age from 21 to 70 years, and had glycosylated hemoglobin levels of 5.8% to 13.9%. METHODS: Four focus groups were held in English and audiotaped. Outreach workers served as translators and comoderators. The content of transcripts was analyzed with Ethnograph software by investigators. The priority issues were confirmed by the comoderators and participants. RESULTS: Participants perceived diabetes as full of complications, emotions, symptoms, and behavior changes. Responses to hyperglycemia were fear, frustration, and uncertainty. Barriers to staying on the prescribed diet were habit, cultural ritual, ideal body image, and limited budget. CONCLUSIONS: Participants suggested that helpful activities would include walking/support group, cooking class, community healthy food store, translated material, and family participation. A community-based diabetes program has been developing as a result of the focus group findings.

How aneuploidy may cause cancer and genetic instability.

It has been difficult to find a common cause for the many and complex phenotypes of cancer such as dedifferentiation, invasiveness, abnormal morphology, growth rate and metabolism, genetic instability, progression to malignancy, cellular heterogeneity of phenotypes and karyotypes, and clonal origin despite heterogeneity. Over 100 years ago aneuploidy, an abnormal balance of chromosomes, was proposed to cause cancer. However, the aneuploidy hypothesis has since been abandoned, in favor of the gene mutation hypothesis, because it could not offer conventional explanations for cancer-specific phenotypes. For example, the aneuploidy hypothesis seemed unable to (i) explain the genesis of abnormal, cancer-specific phenotypes, (ii) reconcile the heterogeneous karyotypes with the clonal origin of cancers, (iii) explain aneuploidy in non-cancerous cells, and (iv) explain how carcinogens would cause aneuploidy. Here we introduce new evidence that aneuploidy offers a simple, coherent explanation of all cancer-specific phenotypes: (i) Congenital and experimental aneuploidy is now known to generate abnormal phenotypes, such as Down syndrome in humans and cancer in animals. (ii) Based on metabolic control analysis, we have derived equations that correlate degrees of aneuploidy with the resulting phenotype abnormalities. These equations suggest that aneuploidy must exceed a certain threshold to generate cancer-specific phenotypes. Therefore, we propose that multistep carcinogenesis corresponds to multiple steps of aneuploidization. (iii) Aneuploidy is also sufficient to explain cancer-specific, karyotypic instability. Since aneuploidy imbalances the highly balance-sensitive components of the spindle apparatus it destabilizes symmetrical chromosome segregation. This autocatalytic instability is the reason why cancers have heterogeneous karyotypes, but are clonal for aneuploidy. Progression to malignancy corresponds to selection of ever more aggressive karyotypic variants. (iv) Both non-genotoxic and genotoxic carcinogens can cause aneuploidy by physical or chemical interaction with mitosis proteins. We conclude that aneuploidy offers a mechanism of phenotype alteration which--above a certain threshold--is sufficient to cause all cancer-specific phenotypes, and is independent of gene mutation.

Diamide-induced cytotoxicity and thermotolerance in CHO cells.

Treatment with the sulfhydryl oxidant diamide denatures and aggregates cellular proteins, which prior studies have implicated as an oxidative damage that activates the heat shock transcription factor and induces thermotolerance. This study was initiated to further characterize cellular response to diamide-denatured proteins, including their involvement in diamide cytotoxicity. Cytotoxic diamide exposures at 37.0 degrees C denatured and aggregated cellular proteins in a manner that was proportional to cell killing, but this correlation was different than that established for heated cells. Diamide exposures at 24.0 degrees C were orders of magnitude less cytotoxic, with little additional killing occurring after diamide was removed and cells were returned to 37.0 degrees C. Thus, protein denaturation that occurred at 37.0 degrees C, after proteins were chemically destabilized by diamide at 24.0 degrees C [Freeman et al., J. Cell. Physiol., 164:356-366 (1995); Senisterra et al., Biochemistry 36: 11002-11011 (1997)], had little effect on cell killing. Thermotolerance protected cells against diamide cytotoxicity but did not reduce the amount of denatured and aggregated protein observed immediately following diamide exposure. However, denatured/aggregated proteins in thermotolerant cells were disaggregated within 17 h following diamide exposure, while no disaggregation was observed in nontolerant cells. This more rapid disaggregation of proteins may be one mechanism by which thermotolerance protects cells against diamide toxicity, as it has been postulated to do against heat killing. As with heat shock, nontoxic diamide exposures induced maximal tolerance against heat killing; however, there was no detectable, increased synthesis of heat shock proteins. Thus, diamide treatment proved to be a reproducible procedure for inducing a phase of thermotolerance that does not require new heat shock protein (HSP) synthesis, without having to use transcription or translation inhibitors to suppress HSP gene expression. These results complement those from studies with other stresses to establish the importance of protein denaturation/aggregation as a cytotoxic consequence of stress and a trigger for thermotolerance induction. The data also illustrate that differences in how proteins are denatured and aggregated can affect their cytotoxicity and the manner in which thermotolerance is expressed.

Genetic instability of cancer cells is proportional to their degree of aneuploidy.

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623-627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells.

Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool-as in speciation-aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

Thermotolerance expression in mitotic CHO cells without increased translation of heat shock proteins.

The objective of this study was to unequivocally demonstrate thermotolerance expression in mammalian cells in the absence of stress-induced synthesis of heat shock proteins (HSPs). Mitotic cells were selected as an experimental system since their genome was in the form of condensed chromosomes and ostensibly incapable of being transcribed; thus, obviating stress-induced HSP gene expression. Asynchronous Chinese hamster ovary (CHO) cells were treated with 0.2 microgram/ml nocodazole to accumulate cells in mitosis for harvest by mitotic shakeoff. Cells were maintained in mitosis with nocodazole during thermotolerance induction, thermotolerance development, and all challenge hyperthermia exposures. Although the heat shock transcription factor was activated by the thermotolerance inducing heat shock, as indicated by gel mobility shift assay, no increase in steady-state HSP mRNA levels was detected, as expected. Preferential synthesis of HSPs from extant mRNA was not detected during thermotolerance development and cellular levels of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins remained constant, as determined by Western Blot analyses. The magnitude and induction threshold of expressed thermotolerance was not diminished when cells were incubated with 10.0 micrograms/ml cycloheximide during thermotolerance development confirming that new protein synthesis was not requisite. Parallel experiments were performed using nonmitotic cells in which protein synthesis was inhibited during thermotolerance development with 10.0 micrograms/ml cycloheximide. As with mitotic cells, high levels of thermotolerance were attained without detectable increases in the cellular content of the 27 kDa, 70 kDa, and 90 kDa heat shock proteins. The results of this study demonstrated that high levels of thermotolerance could be expressed in mitotic cells without stress-induced, preferential synthesis of HSPs, and support the contention that a substantial fraction of thermotolerance expressed in nonmitotic cells also occurs independently of induced HSP synthesis.