[The clinical significance of M proteins].

M protein refers to a monoclonal gammaglobulin that is produced by the monoclonal proliferation of plasma cells. More than 50% of M proteinemia is classified as MGUS, but lymphoproliferative disorders such as multiple myeloma and Waldenström's macroglobulinemia may also develop from MGUS. Therefore, in patients with MGUS it is very important to observe the clinical course. MGUS develops in many disorders including chronic infection, autoimmune disease, various type of neoplasm, neurological disease and skin disease. In general, no treatment is required for MGUS and smoldering type myeloma in which the clinical course and laboratory data are stable. However, in progressive myeloma or cases of plasmacytoma chemotherapy, radiation and/or surgery are indicated. Some factors are known to predict the prognosis of multiple myeloma, and poly(A) polymerase may be a useful indicator for predicting the prognosis of multiple myeloma.

Identification of a novel isoform of poly(A) polymerase, TPAP, specifically present in the cytoplasm of spermatogenic cells.

We have identified cDNA clones encoding a testis-specific poly(A) polymerase, termed TPAP, a candidate molecule responsible for cytoplasmic polyadenylation of preexisting mRNAs in male haploid germ cells. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contained 642 residues, and shared a high degree of identity (86%) with that of a nuclear poly(A) polymerase, PAP II. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAP II. Recombinant TPAP produced by an in vitro transcription/translation system was capable of incorporating the AMP moiety from ATP into an oligo(A)(12) RNA primer in the presence of MnCl(2). Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognized a 70-kDa protein in the cytoplasm of spermatogenic cells. These results suggest that TPAP may participate in the additional extension of mRNA poly(A) tails in the cytoplasm of male germ cells, and may play an important role in spermiogenesis, probably through the stabilization of mRNAs.

Anticancer drug action on poly(A) polymerase activity and isoforms during HeLa and WISH cell apoptosis.

Poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes mRNA polyadenylation. Its activity and isoform levels vary during cell cycle transformation and apoptosis. It has become widely accepted that cell death after DNA damage by anticancer agents is primarily the result of apoptosis and that cells able to evade apoptosis will be resistant to cell killing. The therapeutic agents interferon (IFN), 5-fluorouracil (5-FU) and tamoxifen (Tam) with different mechanisms of action mediate both partial dephosphorylation and inactivation of PAP, detected by immunoblotting analysis and PAP enzyme assay, respectively. We examined the apoptotic tendencies of HeLa and WISH cell lines caused by one of the drugs used, 5-FU. The trend in the cells examined, observed by DAPI and/or DNA fragmentation assay, was found to be accompanied by and reversibly related to PAP activity levels and PAP lower mobility phosphorylated forms of 106 and 100 kDa isoforms. Moreover, a cell type-modulated, differential response of HeLa (chemosensitive cells) versus WISH (drug-resistant diploid cells) has been revealed. This finding yields information on the possible use of PAP as a tumor marker involved in cell commitment and/or induction of apoptosis and may help to improve our understanding of tumor cell sensitivity to anticancer agents.

An intronless gene encoding a poly(A) polymerase is specifically expressed in testis.

Previous work demonstrated that a single pre-mRNA could generate multiple forms of mammalian poly(A) polymerase mRNAs by alternative splicing or alternative polyadenylation. A cDNA encoding a testis-specific poly(A) polymerase was isolated in this study. The transcription level of Papt in testis of a 2 weeks old mouse was much lower than that of the general poly(A) polymerase gene, Pap. However, the transcription ratio of Papt to Pap was reversed in testis of a 4 weeks old mouse. Transient expression analysis showed that GFP-Papt fusion protein is present both in the nucleus and cytoplasm of HeLa cells. These results suggest that Papt is involved in polyadenylation of transcripts expressed during spermatogenesis.

A subset of poly(A) polymerase is concentrated at sites of RNA synthesis and is associated with domains enriched in splicing factors and poly(A) RNA.

We have performed a detailed study of the spatial distribution of a set of mRNA 3' processing factors in human T24 cells. A key enzyme in RNA 3' processing, poly(A) polymerase (PAP), was found in the cytoplasm and throughout the nucleus in a punctated pattern. A subset of the various isoforms of PAP was specifically concentrated at sites of RNA synthesis in the nucleoplasm. Additionally, the other factors necessary for RNA 3' processing, such as CstF, CPSF, and PABII, were also found at these transcription sites. Our data show that the set of 3' processing factors that are presumed to be necessary for most RNA 3' cleavage and polyadenylation is indeed found at sites of RNA synthesis in the nucleoplasm. Furthermore, sites of RNA synthesis that are particularly enriched in both PAP and PABII are found at the periphery of irregularly shaped domains, called speckles, which are known to contain high concentrations of splicing factors and poly(A) RNA. Disruption of RNA 3' processing by the drug 9-beta-D-arabinofuranosyladenine caused the speckles to break up into smaller structures. These findings indicate that there is a spatial and structural relationship between 3' processing and the nuclear speckles. Our studies reveal a complex and distinct organization of the RNA 3' processing machinery in the mammalian cell nucleus.

Polyadenylate polymerases from normal and cancer cells and their potential role in messenger RNA processing: a review.

Two structurally and immunologically distinct species of nuclear polyadenylate [poly(A)] polymerases have been characterized. One of these enzymes is relatively absent in normal tissues but is predominant in primary and transplanted tumors and transformed cell lines. The presence of the tumor type enzyme in fetal liver, but not in regenerating liver, suggests that it is an oncofetal protein. Antibodies against the tumor-type poly(A) polymerases are present in the sera of rats bearing tumors and in some cancer patients. These antibodies are also found in the sera of rats fed hepatocarcinogen even before preneoplastic nodules were visible, which suggests that elicitation of these antibodies is an early event in neoplastic transformation. Autoantibodies against both liver-type and tumor-type poly(A) polymerase are also present in some rheumatic autoimmune sera. Polyclonal antibodies against purified enzyme from a rat hepatoma, which exhibit a single band upon immunoblot analysis, were used in cell-free extracts to study the role of poly(A) polymerase in the 3'-end processing of pre-mRNA. These studies showed that the antibodies blocked both endonucleolytic cleavage and poly(A) addition at the cleavage site and complex formation between factors in the extract and pre-mRNA. Independent studies in other laboratories have demonstrated that both the cleavage and poly(A) polymerase activities require the same component for their function. These observations suggest that both cleavage and polyadenylation reactions are tightly coupled in a functional complex.

Nuclear envelope glycoprotein with poly(A) polymerase activity of rat liver: isolation, characterization, and immunohistochemical localization.

A protein with poly(A) polymerase activity has been identified and isolated from hepatic nuclear envelopes of rats to near homogeneity. The ability of the enzyme to bind to concanavalin A-agarose and to be eluted from the column with methyl alpha-D-mannopyranoside (0.2 M) as well as the inhibitory effects of alpha-mannosidase suggested that it was a glycoprotein. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP, and an oligonucleotide primer. The enzyme activity with Mn2+ was about 20-fold higher than that with Mg2+. Several known inhibitors adversely affected poly(A) polymerase activity. The enzyme has a molecular weight of 64,000 when analyzed by polyacrylamide gel electrophoresis under denaturing conditions and has a sedimentation coefficient of 4.5 S. Immunohistochemical studies using polyclonal antibodies raised against the purified enzyme revealed that the antigen was localized in the nuclear membranes.

Polyadenylate polymerase activity in stationary and growing cell cultures.

Soluble polyadenylic acid (poly(A] polymerase content of stationary and growing cell populations from a variety of cell lines was determined. Cell populations from stationary cultures presented poly(A) polymerase values with a mean of 31 +/- 12 enzyme units/mg protein. The mean value for growing cell populations were 62 +/- 18 enzyme units per mg protein. A statistically significant difference was found between stationary and growing cell populations from the variety of cell lines examined (p less than 0.1). The observed differences in poly(A) polymerase levels persisted after fractionation of the crude extracts and revealed two molecular forms of enzyme activity with a net charge difference in stationary and growing cell cultures.

Structural and immunological identity of rat hepatoma and fetal liver nuclear poly(A) polymerase.

Poly(A) polymerase was partially purified from isolated nuclei of fetal rat liver. Antibodies produced in rabbits immunized with purified nuclear poly(A) polymerase from a rat hepatoma exhibited nearly identical affinity for the partially purified fetal liver and hepatoma enzymes. The extent of the antibody reaction with adult liver nuclear poly(A) polymerase partially purified in a similar manner was only 1.4% of that obtained with the hepatoma enzyme. Immune complex formation was observed between the antibodies and a major polypeptide in the fetal liver enzyme preparation which corresponded to the hepatoma enzyme (mol. wt. 48 000). No other polypeptide in the fetal liver enzyme preparation reacted with the antibodies. The 48-kDa fetal liver polypeptide produced a CNBr cleavage pattern identical to that of hepatoma poly(A) polymerase which is known to be different from the cleavage pattern of the adult liver major nuclear poly(A) polymerase. A fetal liver polypeptide corresponding to the adult liver enzyme (mol. wt. 38 000) was not evident. These results coupled with other data suggest that the hepatoma nuclear poly(A) polymerase is an oncofetal protein.

Structurally and immunologically distinct poly(A) polymerases in rat liver. Occurrence of a tumor-type enzyme in normal liver.

Poly(A) polymerases purified from rat liver nuclei consisted of two distinct species, a predominant enzyme of Mr = 38,000 and a minor one of Mr = 48,000. Prior to extensive purification, the minor enzyme constituted approximately 1% of the total liver poly(A) polymerase. Poly(A) polymerase purified from a rat tumor, Morris hepatoma 3924A, was comprised of a single species of Mr = 48,000 which was identical to the minor liver enzyme with respect to chromatographic and immunological characteristics. Gel filtration on Sephacryl S-200 using 0.3 M NaCl for elution showed that the major liver poly(A) polymerase had a molecular weight of 156,000, which corresponded to a tetramer of the 38-kDa polypeptide, whereas the hepatoma and minor liver 48-kDa species existed as dimers with a molecular weight of 96,000. Fractionation by Sephacryl S-200 resulted in complete loss of both liver poly(A) polymerase activities which could be restored by exogenous N1-type protein kinase. Following CNBr cleavage, the 48-kDa poly(A) polymerase from liver and hepatoma exhibited nearly identical peptide maps which were distinct from that of the major liver enzyme (38 kDa). Antibodies raised against tumor poly(A) polymerase reacted with the 48-kDa polypeptide but not with the 38-kDa liver enzyme. Immune complex formation was observed between seven of the eight CNBr cleavage products derived from the 48-kDa polypeptide of both liver and hepatoma. It is concluded that distinct genes in rat liver code for two structurally and immunologically unique nuclear poly(A) polymerases, one of which is identical to the enzyme from the hepatoma.

[Separate extraction of 2 fractions of Mn++-dependent poly-A polymerase activity in liver cell nuclei of rats]. / Estrazione differenziata di due attivita' poli(A) polimerasiche Mn++-dipendenti dai nuclei di cellule di fegato di ratto.

Part of the Mn++-dependent poly(A) polymerase activity of rat liver nuclei leaks out of the organelles when they are suspended in isotonic sucrose. With 0.5 ml/g fresh tissue of medium or more no increase in enzyme leaking out occurs after 30 min. Three repeated isotonic suspensions yelded all the activity thus obtainable (about 25% of total). Increasing the ionic strength of the suspension medium causes dramatic inactivation of the residual activity both in the extracted fraction and in the remaining one. It is confirmed that the bound activity is attached to chromatin.