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Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.

Purpose: To address the widespread concern that false-positive results during breast MRI screening may have adverse psychological effects., Methods: Impact of Event Scale measurements in 103 high-risk women enrolled in a longitudinal MRI screening study and comparison of subjects with normal results vs. those with prior recall events., Results: Of 189 MRI scans performed, 64 (34%) prompted further evaluation. Subjects with previously abnormal results had significantly higher Avoidance scores at the time of their second MRI. Multivariate analysis showed this was driven by the greater number of BRCA1/2 carriers in that group but was not related to screening recall., Conclusions: Practitioners' concerns about the high false positive rate of breast MRI may not be matched by actual psychological effects in most high-risk women.

Mechanical unfolding of polyproteins by force spectroscopy provides valuable insight into their free energy landscapes. Most experiments of the unfolding process have been fit to two-state and/or one dimensional models, with the details of the protein and its dynamics often subsumed into a zero-force unfolding rate and a distance x{u}{1D} to the transition state. We consider the entire phase space of a model protein under a constant force, and show that x{u}{1D} contains a sizeable contribution from exploring the full multidimensional energy landscape. This effect is greater for proteins with many degrees of freedom that are affected by force; and surprisingly, we predict that externally attached flexible linkers also contribute to the measured unfolding characteristics.

The equilibrium free energy difference between two long-lived molecular species or "conformational states" of a protein (or any other molecule) can in principle be estimated by measuring the work needed to shuttle the system between them, independent of the irreversibility of the process. This is the meaning of the Jarzynski equality (JE), which we test in this paper by performing simulations that unfold a protein by pulling two atoms apart. Pulling is performed fast relative to the relaxation time of the molecule and is thus far from equilibrium. Choosing a simple protein model for which we can independently compute its equilibrium properties, we show that the free energy can be exactly and effectively estimated from nonequilibrium simulations. To do so, one must carefully and correctly determine the ensemble of states that are pulled, which is more important the farther from equilibrium one performs simulations; this highlights a potential problem in using the JE to extract the free energy from forced unfolding experiments. The results presented here also demonstrate that the free energy difference between the native and denatured states of a protein measured in solution is not always equal to the free energy profile that can be estimated from forced unfolding simulations (or experiments) using the JE.

Proteins are actively unfolded to pass through narrow channels in macromolecular complexes that catalyze protein translocation and degradation. Catalyzed unfolding shares many features that characterize the mechanical unfolding of proteins using the atomic force microscope (AFM). However, simulations of unfolding induced by the AFM and when a protein is translocated through a pore suggest that each process occurs by distinct pathways. The link, if any, between each type of unfolding, therefore, is not known. We show that the mechanical unfolding energy landscape of a protein, obtained using an atomistic molecular model, can be used to predict both the relative mechanical strength of proteins when unfolded using the AFM and when unfolded by translocation into a pore. We thus link the two processes and show that the import rate through a pore not only depends on the location of the initiation tag but also on the mechanical properties of the protein when averaged over all the possible geometries that are relevant for a given translocation initiation site.

Single-molecule experiments and their application to probe the mechanical resistance and related properties of proteins provide a new dimension in our knowledge of these important and complex biological molecules. Single-molecule techniques may not have yet overridden solution experiments as a method of choice to characterize biophysical and biological properties of proteins, but have stimulated a debate and contributed considerably to bridge theory and experiment. Here we demonstrate this latter contribution by illustrating the reach of some theoretical findings using a solvable but nontrivial molecular model whose properties are analogous to those of the corresponding experimental systems. In particular, we show the relationship between the thermodynamic and the mechanical properties of a protein. The simulations presented here also illustrate how forced and spontaneous unfolding occur through different pathways and that folding and unfolding rates at equilibrium cannot in general be obtained from forced unfolding experiments or simulations. We also study the relationship between the energy surface and the mechanical resistance of a protein and show how a simple analysis of the native state can predict much of the mechanical properties of a protein.

Recent experiments have demonstrated that proteins unfold when two atoms are mechanically pulled apart, and that this process is different to when heated or when a chemical denaturant is added to the solution. Experiments have also shown that the response of proteins to external forces is very diverse, some of them being "hard," and others "soft." Mechanical resistance originates from the presence of barriers on the energy landscape; together, experiment and simulation have demonstrated that unfolding occurs through alternative pathways when different pairs of atoms undergo mechanical extension. Here we use simulation to probe the mechanical resistance of six structurally diverse proteins when pulled in different directions. For this, we use two very different models: a detailed, transferable one, and a coarse-grained, structure-based one. The coarse-grained model gives results that are surprisingly similar to the detailed one and qualitatively agree with experiment; i.e., the mechanical resistance of different proteins or of a single protein pulled in different directions can be predicted by simulation. The results demonstrate the importance of pulling direction relative to the local topology in determining mechanical stability, and rationalize the effect of the location of importation/degradation tags on the rates of mitochondrial import or protein degradation in vivo.

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.

In this paper we present a new and possibly more effective way of inhibiting thymidylate synthase (TS) in cells than through the use of substrate analogue inhibitors. An inactive double mutant of TS (DM), Arg(126)Glu/Cys(146)Trp, is shown to progressively impair the reactivation of native Escherichia coli TS when the two are denatured together in vitro. The individual single mutant proteins Arg(126)Glu and Cys(146)Trp showed little or no inhibition. When the DM is introduced into E. coli and induced from an expression plasmid, the mutant subunits act as a decoy in deceiving newly formed native TS subunits to fold with them to yield inactive heterodimers. As a consequence of the depletion of TS, the cells die a "thymineless" death when grown in medium devoid of thymine. Addition of thymine to the medium enables the cells to grow normally, although only very low levels of TS activity could be detected in those cells containing induced DM. The individual single-site mutations of the DM, Arg(126)Glu and Cys(146)Trp, did not inhibit growth, as might be expected from the in vitro studies. However, when a nontoxic level of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) is added to growing DM-transformed cells, the combination is lethal to the cells. These experiments suggest that a similar dominant-negative response to the DM of TS could be affected in tumor cells, for which preliminary evidence is presented. This technique, either alone or combined with other modalities, suggest a new approach to targeting cells for chemotherapy.

The phage T4 thymidylate synthase (td) gene contains an intron open reading frame that encodes a 245-amino acid-long basic protein (Chu, F. K., Maley, G. F., West, D. K., Belfort, M., and Maley, F. (1986) Cell 45, 157-166). The open reading frame (Irf) has been cloned as a fusion protein behind a phage T7 promoter and overexpressed in Escherichia coli. The amplified Irf protein is associated with insoluble inclusion bodies and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis about 7 kDa smaller than expected. Data obtained from DNA sequencing, amino acid sequencing of the fusion protein, and carboxypeptidase Y digestion suggest that although the cloned gene is not altered and the protein is made from the expected start codon, it appears to terminate about 90 amino acids before the encoded stop codon. Proteolytic cleavage during or soon after synthesis appears to be responsible for the truncated Irf. The expressed protein is solubilized in guanidine HCl and renatured by dialysis against high salt. This partially purified preparation has been found to contain a DNA endonuclease activity specific for the td delta I gene, which contains a precise deletion of the intron.

Chick embryo cells respond to interferon by producing very high levels of 2-5A synthetase. From inhibitor studies, it appears that the response involves the controlled derepression of an interferon-inducible gene. Continuous interaction of the cells with interferon seems to be necessary to maintain maximal enzyme induction. Partial purification of the synthetase indicates that it is probably composed of a single polypeptide of 56,000 MW. Upon activation by binding to dsRNA, this polypeptide catalyzes a nucleotidyl transferase reaction in which 5'-AMP residues are transferred from ATP to the 2' position of appropriate acceptors. A number of naturally occurring small molecules (in addition to 2-5A itself) that can function as adenylate acceptors have been identified, but the physiological significance of these adenylylation reactions remain to be established.

A newborn 2.4-kg baby girl with a prenatal diagnosis of sacrococcygeal teratoma by ultrasound had a soft cystic mass extending from the infracoccygeal region into the right buttock. The cyst contained meconium leading to a large retroperitoneal cavity. The cyst was drained through an extended buttock incision, and healed without a site of bowel perforation ever identified. Meconium pseudocyst should be included in the differential diagnosis of sacrococcygeal and buttock tumors in the newborn.

Treatment of primary cultures of chicken embryo cells with homologous interferon results in a substantial increase in the level of 2',5'-oligoadenylate synthetase activity that can be detected in cell extracts. This increase can be prevented by inhibitors of RNA or protein synthesis and is thus thought to represent the induction of an interferon-inducible gene, perhaps the 2',5'-oligoadenylate synthetase gene itself. To examine this response in greater detail, we studied its kinetics under the following conditions: (i) cessation of interferon treatment after different lengths of time, (ii) delayed inhibition of RNA or protein synthesis, and (iii) combinations of these treatments. The results showed that in cells treated continuously with interferon, the enzyme level reached a peak after 9 h of treatment and then decreased with a half-life of about 30 h, despite the continued presence of interferon. Removal of interferon during induction reduced the peak level of activity that was attained and somewhat accelerated its decline but did not otherwise affect the time-course of the response. On the other hand, removal of interferon after maximum induction clearly accelerated the decay of enzyme activity. This process could be delayed by inhibitors of protein synthesis, which effectively stabilized the induced enzyme. This behavior is reminiscent of other inducible enzymes, such as the steroid-induced tyrosine aminotransferase, and suggests that the level of 2',5'-oligoadenylate synthetase, which is also inducible by steroid hormones in some cell types, is subject to similar control mechanisms.

The 1017-bp intron within the cloned phage T4 td gene was deleted by oligonucleotide-directed mutagenesis. Induction of thymidylate synthase activity and mature td mRNA from this intronless construct (pKTd delta I) was compared both in vivo and in vitro with expression from plasmids bearing td genes in which the introns contain either no change (pKTd2), an XbaI linker inserted about 200 nucleotides from the 3'-end (pKTdX-1), or a deletion of two-thirds of the central portion (pKTd delta 1-3). Slightly more synthase accumulated in cells carrying pKTd delta I as compared to the other td genes when induction was performed at 30, 37, or 42 degrees C. Dramatically different results were observed in vitro, where enzyme activity synthesized from pKTd delta I DNA appeared earlier and reached severalfold higher levels than with pKTd2 DNA. In addition, thymidylate synthase expression from pKTdX-1 was impaired relative to pKTd2, while pKTd delta 1-3 accumulated enzyme at levels intermediate between those of pKTd2 and pKTd delta I. Under both in vivo and in vitro conditions, increasing levels of mature td mRNA preceded and paralleled those in enzyme activity for all four plasmids, demonstrating comparable translation of the mRNAs produced. From these results it would appear that the splicing of td RNA is much more efficient in vivo than in vitro, suggesting that other cellular components may facilitate in vivo processing of this intron-containing transcript.

The td gene contains a 735 bp open reading frame within its 1017 bp intron. A 12 nucleotide stretch may form a stable secondary structure with the putative Shine-Dalgarno sequence of the intron open reading frame and thus impair its translation. SP6 RNA polymerase transcripts of the td gene synthesized in vitro at 40 degrees C encompass a 2.7 kb primary transcript, a 1.7 kb mRNA, and a 1 kb intron RNA. The excised intron RNA consisted of linear and cyclized forms. RNAase H studies and resistance of the cyclized intron to linearization by HeLa cell debranching enzyme suggest it to be circular. Self-splicing of isolated td primary transcript occurred only marginally at 28 degrees C, but increased progressively to 50 degrees C, and required the presence of both Mg++ and a guanosine cofactor. An internal guide sequence is evident which may align the 5' splice site with the 3' end, presumably for precise exon ligation.

An oligonucleotide polymerase and a protein kinase which require double-stranded RNA (dsRNA) for activation are induced in HeLa cells by human fibroblast interferon. The polymerase synthesizes a series of oligonucleotides from ATP, whereas the kinase phosphorylates a polypeptide of Mr = 72,000 and the alpha subunit of initiation factor eIF-2. Partially or fully 2'-O-methylated derivatives of poly(inosinic acid) . poly(cytidylic acid) (rIn . rCn) were used to determine the structural requirements of dsRNA in the activation of these two enzymes. While fully methylated polymers failed to activate either enzyme, partially methylated polymers activated the enzymes in specific manners. The activation of the kinase by the rIn . rCn analogues was affected more severely by the level of methylation than was the activation of the polymerase. Moreover, fully methylated analogues blocked the activation of the kinase by rIn . rCn but not the activation of the polymerase. These observations are consistent with a biphasic model for enzyme activation similar to that proposed for interferon induction, which required the recognition of a relatively small region of rIn . rCn as the last step. Differences in the activation of the polymerase and kinase are explicable on the basis of the polymerase requirement for a smaller recognition region of the rIn . rCn duplex than the kinase. Dependence of polymerase activation on the level of methylation shows striking similarities with the interferon inducing activities of these analogues, suggesting a possible relationship between polymerase activation and interferon induction.

Treatment of HeLa cells with human fibroblast interferon results in increased levels of latent protein kinase activity that can be activated by double-stranded RNA (dsRNA). The protein kinase activity in extracts of interferon-treated cells is assayed by two methods: (a) inhibition of protein synthesis in rabbit reticulocyte lysates and (b) phosphorylation of two polypeptides of Mr 72000 (P1) and 38000 (the eIF-2 alpha subunit of initiation factor 2). When extracts of interferon-treated cells are fractionated by centrifugation at 150000 x g, the protein kinase activity is found in the pellet fraction. The kinase is maximally activated by 0.1 micrograms/ml poly(I) . poly(C). An increase in protein kinase activity is detected after 8 h of treatment with 100 units interferon/ml or after a 17-h treatment with 12.5 units/ml or greater interferon concentrations. Therefore, the kinase activity increases as a function of both time of treatment and interferon concentration. Addition of actinomycin D to cells during interferon treatment prevents this increase. The protein kinase activity decreases gradually over three days when interferon-treated cells are subsequently grown in the absence of interferon.

This survey examined quality assurance practice in matched pairs of rural and urban hospital clinical laboratories. Questions directed at personnel qualifications, laboratory management, internal quality control mechanisms, and proficiency testing enrollment revealed no major differences between the rural-urban pairs. Deficiencies observed were either generic or size-related. Most laboratories were directed by physicians; however, almost half spent 5 or less hours per week in the laboratory. All respondents employed at least one individual professionally certified at the "technologist" level. Virtually all respondents insisted they employ a laboratory management system for ensuring quality performance, although 20% did not document their practice. Internal quality control practice varied widely. Many respondents recognized shortcomings related to volume and budget, and requested specific training courses for technical personnel. At least 20% of the laboratories were not enrolled in any recognized proficiency testing program, an integral part of the total quality assurance process.