Oncogenic role of DDX3 in breast cancer biogenesis.

Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expressed, we found consistent activation of DDX3, a member of the DEAD box RNA helicase family. Overexpression of DDX3 in MCF 10A cells induced an epithelial-mesenchymal-like transformation, exhibited increased motility and invasive properties, and formed colonies in soft-agar assays. Besides the altered phenotype, MCF 10A-DDX3 cells repressed E-cadherin expression as demonstrated by both immunoblots and by E-cadherin promoter-reporter assays. In addition, an in vivo association of DDX3 and the E-cadherin promoter was demonstrated by chromatin immunoprecipitation assays. Collectively, these results demonstrate that the activation of DDX3 by BPDE, can promote growth, proliferation and neoplastic transformation of breast epithelial cells.

Percutaneous cryoablation of porcine kidneys with magnetic resonance imaging monitoring.

PURPOSE: We determined the feasibility of a percutaneous approach using magnetic resonance imaging (MRI) for creating cryoablation lesions in the porcine kidney. METHODS AND METHODS: Three domestic swine underwent renal cryoablation under MRI guidance with a total of 6 cryoablation lesions created in 5 kidneys. A 3 mm. cryoprobe was placed under MRI guidance using an interventional MRI unit. With a pressurized argon gas cooling unit the cryoablation lesion was created and monitored by MRI. Gross and histological examination of the kidneys was performed 1 week after the procedure. RESULTS: All animals survived the procedure without difficulty. A total of 6 cryoablation lesions were produced in 5 kidneys. The lesions were 1.9 x 1.3 to 3.9 x 1.9 cm. on MRI. Histological examination 1 week after treatment showed that the lesions were 1.7 x 1.0 to 3.2 x 1.2 cm. There was an area of coagulation necrosis surrounded by a transition zone of inflammatory reaction a mean of 0.5 cm. in diameter with each lesion. CONCLUSIONS: Percutaneous renal cryoablation using MRI imaging proved to be a successful technique for guiding probe placement and monitoring ice ball formation. Because MRI allows imaging in 3 planes, it may be useful for cryoablation of intraparenchymatous tumors.

Prospective study of correlations between biopsy-detected high grade prostatic intraepithelial neoplasia, serum prostate specific antigen concentration, and race.

BACKGROUND: High grade prostatic intraepithelial neoplasia (HGPIN), a premalignant lesion of the prostate gland, is more common in black men than in white men. The influence of HGPIN on the serum prostate specific antigen (PSA) concentration is controversial, and correlations between HGPIN and PSA in black men and white men have not been investigated. METHODS: Between January 1992 and December 1998, 411 black men and 639 white men with suspected prostate carcinoma underwent an initial benign prostate biopsy at a single medical center. The presence or absence of HGPIN in the biopsy specimens was determined by one uropathologist. RESULTS: HGPIN was identified in 8.9% of the specimens. When stratified by PSA concentration (< 4.0 ng/mL, 4.0-9.9 ng/mL, and > or = 10.0 ng/mL), HGPIN was associated with an increased PSA concentration only among men with PSA concentrations < 4.0 ng/mL (P = 0.01). The prevalence of HGPIN in the black and white patients was 13.4% and 5.9%, respectively (P < 0.0001), and was significantly greater in black men than in white men with PSA concentrations < 4.0 ng/mL (P = 0.002). Among the patients with PSA concentrations < 4.0 ng/mL, black race was an independent predictor of an increased PSA concentration when adjusted for patient age, prostate volume, and the presence or absence of HGPIN (P = 0.03). CONCLUSIONS: HGPIN is more common in black men than in white men and may produce an increase in the PSA concentration. However, racial differences in the prevalence of HGPIN may not contribute to racial differences in PSA concentrations among men with no clinical or histologic evidence of carcinoma.

Programmed +1 frameshifting stimulated by complementarity between a downstream mRNA sequence and an error-correcting region of rRNA.

Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3). The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs. We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codonoanticodon interaction in the P site. This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA. A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold. Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting. Its function depends on strict spacing from the site of frameshifting. Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons. Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome.

Prostate cancer detection in Black and White men with abnormal digital rectal examination and prostate specific antigen less then 4 ng./ml.

PURPOSE: Prostate cancer is more common in black than in white American men. Experience in a longitudinal prostate cancer screening program implies that cancer detection is greater in black than in white men with an abnormal digital rectal examination and prostate specific antigen (PSA) less than 4 ng./ml. We investigated potential racial differences in cancer detection in men treated in clinical practice who had an abnormal digital rectal examination and PSA less than 4 ng./ml. MATERIALS AND METHODS: Between January 1992 and December 1999 prostate biopsy was done at a Veterans Affairs Medical Center in 179 black and 357 white men with an abnormal digital rectal examination, PSA less than 4 ng./ml. and no history of prostate surgery. Significant racial differences in demographic and clinical parameters were limited to PSA, which was higher in black men (p = 0.01). RESULTS: Cancer was detected in 38 black (21%) and 65 white (18%) men (p = 0.42). There were no significant racial differences in the PSA adjusted cancer detection rate or in the Gleason score of detected disease. In men with PSA less than 1.0, 1.0 to 1.9, 2.0 to 2.9 and 3.0 to 3.9 ng./ml. the detection rate was 4%, 15%, 27% and 29%, respectively. CONCLUSIONS: In clinical practice prostate cancer detection appears to be equivalent in black and white men when an abnormal digital rectal examination is the only indication of malignancy.

Nonsense-mediated decay mutants do not affect programmed -1 frameshifting.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

Translational frameshifting: implications for the mechanism of translational frame maintenance.

The ribosome rapidly translates the information in the nucleic sequence of mRNA into the amino acid sequence of proteins. As with any biological process, translation is not completely accurate; it must compromise the antagonistic demands of increased speed and greater accuracy. Yet, reading-frame errors are especially infrequent, occurring at least 10 times less frequently than other errors. How do ribosomes maintain the reading frame so faithfully? Geneticists have addressed this question by identifying suppressors that increase error frequency. Most familiar are the frameshift suppressor tRNAs, though other suppressors include mutant forms of rRNA, ribosomal proteins, or translation factors. Certain mRNA sequences can also program frameshifting by normal ribosomes. The models of suppression and programmed frameshifting describe apparently quite different mechanisms. Contemporary work has questioned the long-accepted model for frameshift suppression by mutant tRNAs, and a unified explanation has been proposed for both phenomena. The Quadruplet Translocation Model proposes that suppressor tRNAs cause frameshifting by recognizing an expanded mRNA codon. The new data are inconsistent with this model for some tRNAs, implying the model may be invalid for all. A new model for frameshift suppression involves slippage caused by a weak, near-cognate codon.anticodon interaction. This strongly resembles the mechanism of +1 programmed frameshifting. This may mean that infrequent frameshift errors by normal ribosomes may result from two successive errors: misreading by a near-cognate tRNA, which causes a subsequent shift in reading frame. Ribosomes may avoid phenotypically serious frame errors by restricting apparently innocuous errors of sense.

Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift.

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease.

How translational accuracy influences reading frame maintenance.

Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from a second error occurring after recruitment of an errant aminoacyl-tRNA, either spontaneous dissociation causing premature termination or translational frameshifting. This is clearest in programmed translational frameshifting where the mRNA programs errant reading by a near-cognate tRNA; this error promotes a second frameshifting error (a dual-error model of frameshifting). The same mechanism can explain frameshifting by suppressor tRNAs, even those with expanded anticodon loops. The previous model that suppressor tRNAs induce quadruplet translocation now appears incorrect for most, and perhaps for all of them. We suggest that the 'spontaneous' tRNA-induced frameshifting and 'programmed' mRNA-induced frameshifting use the same mechanism, although the frequency of frameshifting is very different. This new model of frameshifting suggests that the tRNA is not acting as the yardstick to measure out the length of the translocation step. Rather, the translocation of 3 nucleotides may be an inherent feature of the ribosome.

Near-cognate peptidyl-tRNAs promote +1 programmed translational frameshifting in yeast.

Translational frameshifting is a ubiquitous, if rare, form of alternative decoding in which ribosomes spontaneously shift reading frames during translation elongation. In studying +1 frameshifting in Ty retrotransposons of the yeast S. cerevisiae, we previously showed that unusual P site tRNAs induce frameshifting. The frameshift-inducing tRNAs we show here are near-cognates for the P site codon. Their abnormal decoding induces frameshifting in either of two ways: weak codon-anticodon pairing allows the tRNA to disengage from the mRNA and slip +1, or an unusual codon-anticodon structure interferes with cognate in-frame decoding allowing out-of-frame decoding in the A site. We draw parallels between this mechanism and a proposed mechanism of frameshift suppression by mutant tRNAs.

A new model for phenotypic suppression of frameshift mutations by mutant tRNAs.

According to the prevailing model, frameshift-suppressing tRNAs with an extra nucleotide in the anticodon loop suppress +1 frameshift mutations by recognizing a four-base codon and promoting quadruplet translocation. We present three sets of experiments that suggest a general alternative to this model. First, base modification should actually block such a four-base interaction by two classical frameshift suppressors. Second, for one Salmonella suppressor tRNA, it is not mutant tRNA but a structurally normal near cognate that causes the +1 shift in-frame. Finally, frameshifting occurs in competition with normal decoding of the next in-frame codon, consistent with an event that occurs in the ribosomal P site after the translocation step. These results suggest an alternative model involving peptidyl-tRNA slippage at the classical CCC-N and GGG-N frameshift suppression sites.

Effect of frameshift-inducing mutants of elongation factor 1alpha on programmed +1 frameshifting in yeast.

The translational apparatus very efficiently eliminates errors that would cause a spontaneous shift in frames. The probability of frameshifting can be increased dramatically by either cis or trans-acting factors. Programmed translational frameshift sites are cis-acting sequences that greatly increase the frequency of such errors, at least in part by causing a transient translational pause. Pausing during programmed +1 frameshifts occurs because of slow recognition of the codon following the last read in the normal frame. Frameshifting can also be elevated in strains carrying mutations in the homologous elongation factors EF-Tu in bacteria, and EF-1alpha in the yeast Saccharomyces cerevisiae. This phenotype implies that the factors contribute to frame maintenance. Because EF-Tu/EF-1alpha modulate the kinetics of decoding, it is possible that the frameshift suppressor forms of the factors transiently slow normal decoding, allowing spontaneous frameshifting to occur more efficiently, resulting in phenotypic suppression. We have used a set of frameshift reporter plasmids to test the effect of suppressor forms of EF-1alpha on constructs that differ widely in the efficiency with which they stimulate +1 shifting. When these results were compared to the effect of increased translational pausing, it was apparent that the mutations affecting EF-1alpha do not simply prolong the translational pause. Rather, they appear to generally increase the likelihood of frame errors, apparently by affecting the error correction mechanism of the ribosome.

GCR1-dependent transcriptional activation of yeast retrotransposon Ty2-917.

Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstream and downstream of the transcription initiation site. An upstream activation sequence (UAS) and a downstream enhancer stimulate transcription synergistically. Here we show that activation by both of these sites depends on the GCR1 product, a transcription factor which also regulates the genes encoding yeast glycolytic enzymes. Eliminating GCR1 causes a 100-fold decrease in transcription of Ty2-917. Activation by the isolated Ty2-917 UAS also strongly depends on GCR1. Unexpectedly, GCR1-dependent activation by the Ty2-917 enhancer is strongly position-dependent. Activation by the enhancer in its normal position within the transcription unit depended strongly on GCR1, but eliminating GCR1 reduced activation only three-fold when the enhancer was moved upstream of the transcribed region. Gel mobility shift and DNaseI protection assays indicated that GCR1 binds specifically to multiple sites within the Ty2-917 UAS and enhancer regions.

Programmed translational frameshifting.

Errors that alter the reading frame occur extremely rarely during translation, yet some genes have evolved sequences that efficiently induce frameshifting. These sequences, termed programmed frameshift sites, manipulate the translational apparatus to promote non-canonical decoding. Frameshifts are mechanistically diverse. Most cause a -1 shift of frames; the first such site was discovered in a metazoan retrovirus, but they are now known to be dispersed quite widely among evolutionarily diverse species. +1 frameshift sites are much less common, but again dispersed widely. The rarest form are the translational hop sites which program the ribosome to bypass a region of several dozen nucleotides. Each of these types of events are stimulated by distinct mechanisms. All of the events share a common phenomenology in which the programmed frameshift site causes the ribosome to pause during elongation so that the kinetically unfavorable alternative decoding event can occur. During this pause most frameshifts occur because one or more ribosome-bound tRNAs slip between cognate or near-cognate codons. However, even this generalization is not entirely consistent, since some frameshifts occur without slippage. Because of their similarity to rarer translational errors, programmed frameshift sites provide a tool with which to probe the mechanism of frame maintenance.