No abstract

Since chromosomal instability (CIN) is a hallmark of most cancer cells, it is essential to identify genes whose alteration results into this genetic instability. Using a yeast CIN indicator strain, we show that inactivation of the YMR131c/RRB1 gene, which is involved in early ribosome assembly and whose expression is induced when the spindle checkpoint is activated, alters chromosome segregation and blocks mitosis at the metaphase/anaphase transition. We demonstrate that RRB1 interacts with YPH1 (yeast pescadillo homologue 1) and other members of the Yph1 complex, RPL3, ERB1 and ORC6, involved in ribosome biogenesis and DNA replication. Transient depletion of the human homologues GRWD, Pescadillo, Rpl3, Bop1 and Orc6L resulted in an increase of abnormal mitoses with appearance of binucleate or hyperploid cells, of cells with multipolar spindles and of aberrant metaphase plates. If deregulation of proteins involved in ribosome biogenesis, commonly observed in malignant tumors, could contribute to cancer through an aberrant protein synthesis, our study demonstrates that alteration of proteins linking ribosome biogenesis and DNA replication may directly cause CIN.

Human inter‐α‐trypsin inhibitor is a plasma protein of Mr 180000 which has long been described as a single polypeptide chain. However, we have previously demonstrated that it is synthesized in liver by two different mRNA populations coding for heavy or light polypeptide chains [Bourguignon, J. et al. (1983) FEBS Lett. 162, 379–383] and cDNA clones for the heavy or light chains have recently been isolated and characterized [Bourguignon, J. et al. (1985) Biochem. Biophys. Res. Commun. 131, 1146–1153; Salier, J. P. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8272–8276]. In the present study, we show that human poly(A)‐rich RNAs hybrid‐selected with various heavy‐chain‐encoding cDNA clones translate three different heavy chains, designated H1 (Mr 98000) and H3 (Mr 107000). We previously characterized two heavy‐chain cDNA clones. We now report that they correspond to H1 and H2 chains. We have also determined the sequence of an additional cDNA clone which codes for H3 chain. Its insert size is 1.79 kb with a single open reading frame and a poly(A) tail. The deduced amino acid sequence of the H3 chain is highly similar to those of the H1 (54%) and H2 (44%) chains. Northern analysis of human liver poly(A)‐rich RNAs with the three heavy‐chain cDNAs as probes clearly identified a single major mRNA population of 3.3 ± 0.1 kb. Chromosomal localization by in situ hybridization shows that inter‐α‐trypsin inhibitor genes are located on three different human chromosomes. The H1 and H3 genes are located in the p211–p212 region of chromosome 3, whereas the H2 gene resides in the p15 band of chromosome 10. The light‐chain gene is located in the q32–q33 region of chromosome 9. These results indicate that heavy and light chains of inter‐α‐trypsin inhibitor are encoded by at least four functional genes.

To identify genes which overexpression results into chromosomal instability (CIN), we developed a biological approach based on a yeast indicator strain in which CIN can be detected by a sectoring phenotype. Screening in this strain of a yeast genomic library cloned into a high copy vector led us to identify, among the clones generating 100% of sectoring colonies, Clb5, one of the six B-type cyclins present in yeast. Overexpression of cyclin B2 and cyclin B1, the two human homologs of Clb5, in the CIN indicator strain resulted also into a sectoring phenotype and induced, like overexpression of Clb5, an abnormal sensitivity to benomyl, indicating that overexpression of B-type cyclins alters the spindle checkpoint. In a series of 38 primary colorectal cancers, we detected in ®ve tumors (13%) an accumulation of cyclin B1, which was neither related to mRNA overexpression nor to mutation within the coding region, and in ®ve other tumors (13%) a 2 ± 10-fold increase of cyclin B2 mRNA which was not related to gene ampli®cation. These results suggest that overexpression of cyclins B, resulting from di erent mechanisms, could contribute, through an alteration of the spindle checkpoint, to the chromosomal instability observed in cancer.

Huiman inter-a-trypsin inhibitor (IaTI) is a plasma glycoprotein of Mr 180,000, which has been described as a single polypeptide chain. Recently, however, we proposed that IMTI might be composed of a heavy (H) chain (Mr = 95,000) and a light (L) chain (Mr = 40,000) synthesized by two separate mRNAs. In the present study we have characterized cDNAs for the H chain of Io6TI. These cDNAs collectively covered two sequences (977 and 1450 base pairs in length) with single open reading frames. The deduced amino acid sequences were highly homologous to each other and well matched with partial amino acid sequences obtained from purified serum IaTI. RNA blot analyses of liver RNAs with H-or L-chain cDNAs as probes clearly identified two distinct mRNAs of 3.3 and 1.3 kilobases, which corresponded to H or L chain, respectively. Poly(A)+ RNAs hybrid-selected with H-chain cDNAs coded for polypeptide chains of Mr 90,000-95,000.These results unambiguously establish that IaTI is made of multipolypeptides, possibly including one H and two L chains. The H chain contains potential calcium-binding sites and also regions homologous to the proposed reactive site for thiolproteinase inhibitors. These data indicate that IaTI is a complex, multifunctional protein. mRNAs for both the H and L chains were found only in liver.

Among the identified factors involved in malignant transformation, abnormal methylation of the CDKN2A/p16 INK4a gene promoter has been described as an early event, particularly in bronchial cell cancerization. Precancerous bronchial lesions (n ‫؍‬ 70) prospectively sampled during fluorescence endoscopy in a series of 37 patients at high risk for lung cancer were studied with respect to the methylation status of the CDKN2A gene. Methylation-specific polymerase chain reaction was performed on DNA extracted from pure bronchial cell populations derived from biopsies and detection of p16 protein was studied by immunohistochemistry on contiguous parallel biopsies. Aberrant methylation of the CDKN2A gene promoter was found in 19% of preinvasive lesions and its frequency increased with the histologic grade of the lesions. Methylation in at least 1 bronchial site was significantly more frequent in patients with cancer history, although there was no difference in the outcome of patients with or without methylation in bronchial epithelium. The other risk factors studied (tobacco and asbestos exposure) did not influence the methylation status. There was no relationship between CDKN2A methylation and the evolutionary character of the lesions. Our results confirm that abnormal methylation of the CDKN2A gene promoter is an early event in bronchial cell cancerization, which can persist for several years after carcinogen exposure cessation, and show that this epigenetic alteration cannot predict the evolution of precancerous lesions within a 2-year follow-up. © 2002 Wiley-Liss, Inc.Key words: promoter methylation; bronchial biopsies; CDKN2A/ p16 INK4a ; NSCLC Carcinogenesis of the respiratory epithelium is thought to require multiple steps, with premalignant epithelial modifications preceding invasive cancers. 1 In squamous cell carcinoma of the bronchus, a model of progression from premalignant lesions to invasive cancer has been proposed that includes the sequential development of basal cell hyperplasia, squamous metaplasia, mild to moderate and severe dysplasia and carcinoma in situ (CIS). 2 This sequence has regularly been observed in animal models of carcinogenesis (reviewed in ref.3) and supported by extensive postmortem examination of the bronchial tree from smokers and lung cancer patients. 4 As in other epithelial cancers such as colon cancer, each step of the bronchial carcinogenesis is thought to be the result of the accumulation of genetic and epigenetic damages involving several oncogenes and tumor suppressor genes. 5 Among the molecular alterations frequently detected in lung cancers, inactivation of the RB1 pathway is a common event that allows tumoral cells to escape from the G1/S transition checkpoint. Loss of the G1 cyclindependent kinase inhibitor 2A (CDKN2A/p16 INK4a ) and/or overexpression of cyclin D1 are more commonly found in nonsmall cell lung cancer (NSCLC), 6,7 whereas the inactivation of this pathway in small cell lung carcinomas (SCLC) mainly results from a loss of expression of the RB1 protein. 8,9 ...

The human inter-a-trypsin inhibitor (ITI) light-chain gene, which codes for the two proteins a,-microglobulin (protein HC) and ITI-derived human inhibitor of 30 kDa (HI-30), was isolated from a human genomic library. This gene, present as a single copy in the human genome, is composed of 10 exons and 9 introns distributed over 20 kbp. A single transcriptional initiation site was identified in the 5'-flanking region which contained promoter elements, but no typical TATA box. However a sequence equivalent to the TATA box is present on both sense and anti-sense strands in the 5'-flanking region of the first exon coding for HI-30. The exon-intron organization suggests that the regions coding for protein HC and other members of the lipocalin superfamily evolved from a common ancestral gene that is probably different from that coding for HI-30. These data suggest that two distinct ancestral genes could have existed and fused during evolution. Several direct and one inverted repeats are also found within this gene, as well as potential glucorticoid-receptor binding sites.Inter-a-trypsin inhibitor (ITI) is a serine protease inhibitor of 220 kDa present in human plasma. Polypeptides of lower molecular mass are also found in plasma, urine and bronchial mucus ; they display an immunological cross-reactivity with the inhibitor and are called derivatives [l]. For a long time, inter-a-trypsin inhibitor was described as a single polypeptide chain; however, we reported that it is synthesized by separate mRNAs coding for heavy (90 -95 kDa) and light (40 kDa) chains [2]. Two separate IT1 heavy-chain cDNA clone families [3, 41, then a third one [5], have been isolated and sequenced; they displayed highly similar sequences and coded for three heavy polypeptides [5]. cDNAs coding for the light-chain were also isolated [6, 71; the deduced amino acid sequence corresponds to two proteins: a,-microglobin (protein HC) and a 30-kDa derivative of human inter-a-trypsin inhibitor (HI-30). The latter contains two tandem Kunitz-type domains (domains I and 11) with an inhibitor site for leukocyte elastase as well as an inhibitor site for trypsin and chymotrypsin [8, 91. a,-Microglobulin has been characterized as a 31-kDa glycoprotein found in serum, urine and cerebrospinal fluid [lo, 111.

Pre-a-trypsin inhibitor (Pal) is a serine-proteinase inhibitor of M, 130000 found in human serum. This protein belongs to the family of proteins called inter-a-trypsin inhibitor (ITI). Pa1 is composed of a heavy chain (HC3) and of a light chain (bikunin), synthesized by two separate mRNA. Bikunin is identical to the IT1 light chain, the structure of which has already been established. The HC3 is obtained from a precursor called H3. The bikunin is covalently linked to HC3 by a chondroitin-4-sulfate glycosaminoglycan. We report here the H3 full-length cDNA sequence and the deduced amino-acid sequence of the heavy-chain H3 precursor. The high degree of similarity between the nucleotide and amino-acid sequences of IT1 heavy-chain families H1, H2, H3 is examined with respect to their probable structure and assembly with bikunin in the final proteins, Pal and ITI.Pre-a-trypsin inhibitor (PaI) is a recently described glycoprotein of M, 130000 [l], structurally related to the intera-trypsin-inhibitor (ITI) family of serine-proteinase inhibitors. Pal is composed of one heavy-chain of M, 90000, called HC3, and one light chain of M , 30000 named bikunin because it is composed of two pancreatic-trypsin-inhibitor(Kunitz)-type domains [2]. The isolation and sequence of ITI-light-chain cDNA [3, 41 and gene [5] showed that they code for two tandemly arranged proteins : a-1-microglobulin and bikunin. Pal chains are covalently linked to each other by an unusual structure among plasma proteins: the interchain linkage is mediated through esterification of the a-carbon of the C-terminal Asp of HC3 with the C6 of an internal N-acetylgalactosamine of the glycosaminoglycan chain 0-linked to SerlO of the bikunin [6]. The sequence analysis of peptides derived from the proteolysis of Pal indicates that the heavy-chain precursor (heavy-chain precursors are called H, whereas mature heavy chains are denominated HC, according to [l]) of this protein is coded by the IT1 H3 heavy-chain cDNA which we previously described [7]. We have also shown that the H3 gene is located in the p211-p212 region of chromosome 3 [7]. Abbreviations. PaI, pre-a-trypsin inhibitor; ITI, inter-a-trypsin inhibitor; H1, H2 and H3, heavy chain precursor of inter-a-trypsin inhibitor family; HC1, HC2 and HC3, mature heavy chain of inter-atrypsin-inhibitor family; RACE, rapid amplification of cDNA ends.Enzymes. Reverse transcriptase (EC 2.7.7.49) ; DNA polymerase I, Tuq DNA polymerase (EC 2.7.7.7).Note. The novel nucleotide sequence data reported here have been deposited in the EMBL/GenBank library under accession number X 67055.In the present study, we report the complete cDNA sequence of H3 heavy chain, and therefore the complete amino-acid sequence of Pa1 precursor HC3 heavy chain. The amino-acid-sequence similarity between ITI-heavy-chain families H1, H2 and H3 was also studied. MATERIALS AND METHODS ReagentsRestriction endonucleases, reverse transcriptase, M13 mp18, M13 mp19 and pUC 18 vectors were from Bethesda Research Laboratories. Nitrocellulose filters (BA 85) we...