High Ki-67 proliferative index predicts disease specific survival in patients with high-risk soft tissue sarcomas.

BACKGROUND: Soft tissue sarcomas (STSs) are heterogeneous neoplasms that have variable clinical outcome. Several clinical parameters and few molecular markers, including Ki-67 proliferative index, have been shown to correlate with patient prognosis. To the authors' knowledge, no definitive report exists to identify one molecular marker that can be analyzed easily in a clinical setting and that predicts survival in a cohort of patients with high-risk STS of identical clinical characteristics but variable outcome. METHODS: The influence of clinical prognostic factors was eliminated by selecting two patient groups with identical high-risk characteristics: large (> 10 cm), high-grade, deep, completely resected primary extremity STS (n = 47). Patients in the first group remained disease free (no evidence of disease [NED]) after primary tumor treatment (n = 19), whereas patients in the second group subsequently died of disease (DOD; n = 28). Triplicate 0.6-mm core biopsies from defined morphologic areas of paraffin embedded primary tumors were assembled on a tissue microarray and analyzed by immunohistochemistry with the MIB-1 antibody, and Ki-67 proliferative indices were correlated with patient outcome. RESULTS: High Ki-67 proliferative index, defined as greater than 30% tumor cells showing nuclear immunoreactivity, was significantly more frequent in the DOD group than in the NED group and was associated with tumor-related mortality (P = 0.02). This marker identifies an especially aggressive malignant phenotype within a cohort of high-risk tumors that is based on well established clinical and pathologic parameters alone and is easy to use in a clinical setting. CONCLUSIONS: On the basis of these data and previous reports, high Ki-67 proliferative index is suggested as a significant factor for predicting the prognosis of patients with high-risk STS and should be evaluated prospectively based on clinical trials.

Drosophila p53 is a structural and functional homolog of the tumor suppressor p53.

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.

Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems.

In an in-gel polymerase chain reaction (PCR), the generation of a 1750-bp yeast DNA fragment was inhibited when yeast DNA gel-stabs or gel-slices stained with ethidium bromide (EtBr) or SYBR Green I were used. Similar inhibition occurred to a varying degree in the reamplification of PCR fragments in prokaryotic systems. Inclusion of the dyes in PCR resulted in an inhibition at about 10 microg/ml EtBr and at 10,000-20,000-fold dilution of SYBR Green I in all systems. The effect remained unchanged despite increasing the PCR cycles to 40. However, increasing the magnesium chloride concentration did reverse the inhibitory actions, although the PCR specificity was lost. In an unusual observation, we find that, at higher dye concentrations (50 microg/ml EtBr, or thousand fold dilution of SYBR Green I), the input yeast DNA electrophoretic profile is maintained following 25 PCR cycles (despite a denaturation temperature of 94 degrees C). It varied significantly in different DNA systems and was readily reversed by high Mg++ concentrations. It is concluded that, at low Mg++ concentrations, different PCR systems are inhibited to varying extents by intercalating dyes and, in some PCR systems, intercalating dyes at unusually high concentrations maintain input DNA electrophoretic profile.

p73 function is inhibited by tumor-derived p53 mutants in mammalian cells.

The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator. Recent studies have identified a p53 relative, termed p73. We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73alpha and -beta isoforms to transactivate various p53-responsive promoters. We show that both p73alpha and p73beta activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299. However, a number of significant differences were observed between p53 and p73 and even between p73alpha and p73beta. Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37 degreesC. Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay. When we examined complex formation in transfected mammalian cells, we found that p73alpha coprecipitates with mutant but not wild-type p53. Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73. By cotransfecting p73alpha along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73alpha. This decrease in transcriptional activity is correlated with the reduced ability of p73alpha to promote apoptosis in the presence of tumor-derived p53 mutants. Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.

The TOR signaling cascade regulates gene expression in response to nutrients.

Rapamycin inhibits the TOR kinases, which regulate cell proliferation and mRNA translation and are conserved from yeast to man. The TOR kinases also regulate responses to nutrients, including sporulation, autophagy, mating, and ribosome biogenesis. We have analyzed gene expression in yeast cells exposed to rapamycin using arrays representing the whole yeast genome. TOR inhibition by rapamycin induces expression of nitrogen source utilization genes controlled by the Ure2 repressor and the transcriptional regulator Gln3, and globally represses ribosomal protein expression. gln3 mutations were found to confer rapamycin resistance, whereas ure2 mutations confer rapamycin hypersensitivity, even in cells expressing dominant rapamycin-resistant TOR mutants. We find that Ure2 is a phosphoprotein in vivo that is rapidly dephosphorylated in response to rapamycin or nitrogen limitation. In summary, our results reveal that the TOR cascade plays a prominent role in regulating transcription in response to nutrients in addition to its known roles in regulating translation, ribosome biogenesis, and amino acid permease stability.

Human tumor-derived p53 proteins exhibit binding site selectivity and temperature sensitivity for transactivation in a yeast-based assay.

p53 is a sequence-specific transcriptional activator with a number of known target genes which contain p53-responsive elements. Mutations in p53 have been identified within its sequence-specific DNA binding domain in more than half of all human tumors, although a subset of tumor-derived p53 mutants have retained the ability to bind DNA and activate transcription under certain conditions. In order to broaden our understanding of this transactivating ability, we examined the efficacy by which p53 mutants bind to and activate reporters in an Saccharomyces cerevisiae-based assay. Analysis of 19 human tumor-derived p53 mutants, spanning the DNA binding domain of p53 and including the 'hot-spot' class, revealed a broad array of transcriptional transactivation abilities at 24 degrees C, 30 degrees C and 37 degrees C, despite the fact that each mutant had originally been identified as being inactive for transactivation in yeast against a single p53-responsive RGC site-containing reporter. One class of mutants (P177L, R267W, C277Y and R283H) retained wild-type or near wild-type activity that is binding site-selective, even at physiological temperature (37 degrees C). Another class of mutants (V143A, M1601/A161T, H193R, Y220C and 1254F), all positioned for maintaining the beta-scaffold of p53, also retained selective activity, but preferentially at sub-physiological temperatures (24 degrees and 30 degrees C). Strikingly, however, in contrast to the other tumor derived mutants, all of the previously identified 'hot-spot' mutants were completely inactive with all sites tested. Moreover, a double mutant, L22E/W23S, located within the activation region and previously shown to be transcriptionally inactive in fibroblasts, retained wild-type or near wild-type binding site-selective activity in yeast. Finally, we found that transcriptional activity in vivo does not necessarily correlate with DNA binding in vitro.

Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases.

We identified an essential Saccharomyces cerevisiae protein, Tap42, that associates with Sit4, a type 2A-related protein phosphatase, and with the type 2A phosphatase catalytic subunits. The association of Tap42 with the phosphatases does not require the previously identified phosphatase subunits. Genetic analysis suggests that Tap42 functions positively with both phosphatases. Mutations in TAP42 can confer almost complete rapamycin resistance. In addition, Tap42/Sit4 and Tap42/PP2A complex formation is regulated by nutrient growth signals and the rapamycin-sensitive Tor signaling pathway. These findings, combined with the defect in translation of the tap42-11 mutant at the nonpermissive temperature, suggest that Tap42, Sit4, and PP2A are components of the Tor signaling pathway.

The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase.

SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4. Through such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physically associates with SIT4 in separate complexes. The SAPs function positively with SIT4, and by several criteria, the loss of all four SAPs is equivalent to the loss of SIT4. The data suggest that the SAPs are not functional in the absence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs. The SAPs are hyperphoshorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4. By sequence similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group. Overexpression of a SAP from one group does not suppress the defects due to the loss of the other group. These findings and others indicate that the SAPs have distinct functions.

Activation of CLN1 and CLN2 G1 cyclin gene expression by BCK2.

The Saccharomyces cerevisiae CLN3 protein, a G1 cyclin, positively regulates the expression of CLN1 and CLN2, two additional G1 cyclins whose expression during late G1 is activated, in part, by the transcription factors SWI4 and SWI6. We isolated 12 complementation groups of mutants that require CLN3. The members of one of these complementation groups have mutations in the BCK2 gene. In a wild-type CLN3 genetic background, bck2 mutants have a normal growth rate but have a larger cell size, are more sensitive to alpha-factor, and have a modest defect in the accumulation of CLN1 and CLN2 RNA. In the absence of CLN3, bck2 mutations cause an extremely slow growth rate: the cells accumulate in late G1 with very low levels of CLN1 and CLN2 RNA. The slow growth rate and long G1 delay of bck2 cln3 mutants are cured by heterologous expression of CLN2. Moreover, overexpression of BCK2 induces very high levels of CLN1, CLN2, and HCS26 RNAs. The results suggest that BCK2 and CLN3 provide parallel activation pathways for the expression of CLN1 and CLN2 during late G1.

Overexpression of SIS2, which contains an extremely acidic region, increases the expression of SWI4, CLN1 and CLN2 in sit4 mutants.

The Saccharomyces cerevisiae SIS2 gene was identified by its ability, when present on a high copy number plasmid, to increase dramatically the growth rate of sit4 mutants. SIT4 encodes a type 2A-related protein phosphatase that is required in late G1 for normal G1 cyclin expression and for bud initiation. Overexpression of SIS2, which contains an extremely acidic carboxyl terminal region, stimulated the rate of CLN1, CLN2, SWI4 and CLB5 expression in sit4 mutants. Also, overexpression of SIS2 in a CLN1 cln2 cln3 strain stimulated the growth rate and the rate of CLN1 and CLB5 RNA accumulation during late G1. The SIS2 protein fractionated with nuclei and was released from the nuclear fraction by treatment with either DNase I or micrococcal nuclease, but not by RNase A. This result, combined with the finding that overexpression of SIS2 is extremely to a strain containing lower than normal levels of histones H2A and H2B, suggests that SIS2 might function to stimulate transcription via an interaction with chromatin.