Heterogeneous nuclear ribonucleoprotein A2 is a SET-binding protein and a PP2A inhibitor.

The oncoprotein SET participates in a diversity of cellular functions including cell proliferation. Its role on cell cycle progression is likely mediated by inhibiting cyclin B-cdk1 and the protein phosphatase 2A (PP2A). On identifying new SET cellular partners, we found that SET interacts in vitro and in vivo with the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2); a protein involved in various aspects of mRNA biogenesis. The SET-binding region of hnRNPA2 is the RNP1 sequence that belongs to the RNA-binding domain (RBD) of this protein. We also found that hnRNPA2 has much higher affinity for single-standed DNA than for SET. On analysing the effect of hnRNPA2 on PP2A inhibition by SET, we observed that hnRNPA2 cooperates with SET on PP2A inhibition. This is because we found that hnRNPA2 is also a PP2A inhibitor. HnRNPA2 interacts with PP2A by the RNP1 sequence; however, to inhibit PP2A activity, the complete RBD is needed. We also observed that overexpression of hnRNPA2 inhibits PP2A activity and stimulates cell proliferation. Interestingly, the overexpression of the complete RBD is sufficient to stimulate proliferation. As hnRNPA2 is overexpressed in a variety of human tumors, our results suggest that hnRNPA2 might participate in oncogenesis by stimulating cell proliferation.

Glyceraldehyde 3-phosphate dehydrogenase is a SET-binding protein and regulates cyclin B-cdk1 activity.

We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts in vitro and in vivo with the protein SET. This interaction is performed through the acidic domain of SET located at the carboxy terminal region. On analysing the functional relevance of SET-GAPDH interaction, we observed that GAPDH reverses in a dose-dependent manner, the inhibition of cyclin B-cdk1 activity produced by SET. Similarly to SET, GAPDH associates with cyclin B, suggesting that the regulation of cyclin B-cdk1 activity might be mediated not only by the interaction of GAPDH with SET but also with cyclin B. To analyse the putative role of GAPDH on cell cycle progression, HCT116 cells were transfected with a GAPDH expression vector. Results indicate that overexpression of GAPDH does not affect the timing of DNA replication but induces an increase in the number of mitosis, an advancement of the peak of cyclin B-cdk1 activity and an acceleration of cell cycle progression. All these results suggest that GAPDH might be involved in cell cycle regulation by modulating cyclin B-cdk1 activity.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Calmodulin binds to K-Ras, but not to H- or N-Ras, and modulates its downstream signaling.

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.

Nuclear protein patterns in normal T-lymphocytes and lymphoblastoid cells.

We compared the two-dimensional patterns of nuclear proteins obtained from normal quiescent T lymphocytes with those from normal proliferating T lymphocytes and three lymphoblastoid cell lines (CEM, Namalwa, and Molt-4). We identified sets of nuclear proteins which are specific for normal quiescent or normal proliferating T lymphocytes, or shared by the three lymphoblastoid cell lines and absent from the normal T cells. The protein patterns from two nuclear subfractions, i.e., S1 fraction, obtained after nuclease extraction, and the nuclear matrix, were also analyzed. In S1 nuclear fraction, 6 proteins of 75 kDa [isoelectric point (pI) 4.4], 55 kDa (pI 6.7), 41 kDa (pI 4.1), 39 kDa (pI 5.0), 32 kDa (pI 5.5), and 29 kDa (pI 6.6) were found to be specifically present in normal quiescent cells but not in normal proliferating or lymphoblastoid cell lines. Five proteins of 23 kDa (pI 4.2), 23 kDa (pI 4.3), 22 kDa (pI 4.4), 21 kDa (pI 4.5), and 21 kDa (pI 4.6) were observed only in the S1 fraction of normal proliferating lymphocytes, whereas they were absent in normal quiescent cells and in the transformed cell lines. Eight proteins of 56 kDa (pI 4.7), 50 kDa (pI 4.6), 45 kDa (pI 4.4), 43 kDa (pI 4.3), 42 kDa (pI 4.3), 41 kDa (pI 4.3), 43 kDa (pI 4.2), and 42 kDa (pI 4.1) were found only in the nuclear matrix of normal quiescent cells. Moreover, two doublets of proteins of 31-33 kDa (pI 4.3) and 31-33 kDa (pI 4.2) were found only in the nuclear matrix of the normal proliferating cells and three proteins of 37 kDa (pI 3.8), 37 kDa (pI 3.7), and 35 kDa (pI 4.5) were specifically present in the nuclear matrix of the lymphoblastoid cells lines, but not in normal quiescent or activated lymphocytes.

[Lys61]N-Ras is able to induce full activation and nuclear accumulation of Cdk4 in NIH3T3 cells.

The elements of the cell cycle regulatory machinery activated by the oncogenic form of Ras, [Lys61]N-Ras, have been analysed in NIH3T3 cells. We demonstrate that [Lys61]N-Ras expression is able to induce full cdk4 activation. As already reported, oncogenic Ras expression was sufficient to induce cyclin D1 and p21cip1 expression and their association with cdk4. Furthermore, serum-starved [Lys61]N-Ras NIH3T3 cells showed nuclear accumulation of cyclin D1 and cdk4 not observed in serum-starved NIH3T3 cells. This accumulation of cdk4 into the cell nucleus observed in serum-starved [Lys61]N-Ras NIH3T3 cells was inhibited by a microinjection of neutralizing anti-Ras antibodies. Thus, active [Lys61]N-Ras was a sufficient signal to induce nuclear accumulation of cyclin D1/cdk4, leading to its full activation. Transfection of [Lys61]N-Ras NIH3T3 cells with an inactive form of MEK or their treatment with PD 98059, showed that nuclear translocation of cdk4 was MEK dependent. Interestingly, cells constitutively expressing [Lys61]N-Ras did not inactivate pRb and did not proliferate in the absence of serum. This may be due to the fact that although association of cdk2 with cyclin E and the translocation of those complexes to the nucleus were achieved, [Lys61]N-Ras expression was not sufficient to induce cdk2 activation. The high levels of p27(kip1) that were found in cyclin E/cdk2 complexes may be responsible for the inability of oncogenic Ras to activate this kinase. In consequence, oncogenic alterations that lead to a decrease in p27kip1 bound to cyclin E may cooperate with Ras to induce full cdk2 activation, pRb inactivation and thus cell proliferation.

Disruption of the antiproliferative TGF-beta signaling pathways in human pancreatic cancer cells.

Resistance to TGF-beta1 occurred in pancreatic cancer cells suggesting that inactivation of TGF-beta inhibitory signaling pathways may play an important role in human pancreatic cancer. The aim of our study was to determine the presence of alterations in the main putative components of the TGF-beta inhibitory signaling pathways (p15, Smad4, Smad2, TGFbeta-RII, CDC25A). A panel of human carcinomas of the exocrine pancreas orthotopically implanted and perpetuated in nude mice and pancreatic cancer cell lines were studied. p15 gene alterations, mainly homozygous deletions that involved exons 1 and/or 2, were found in the 62.5% (5 of 8) of pancreatic xenografts whereas Smad4 gene aberrations were found in one of eight xenografts and in two of seven cell lines. Additional aberrations in these genes were acquired during in vivo perpetuation and distal dissemination. Paradoxically, TGFbeta-RII overexpression and a decrease in CDC25A protein levels were found in all tumors and cell lines. In one cell line, resistance to TGF-beta1 occurred in the absence of alterations in the genes analysed so far. We conclude that all human pancreatic tumor cells analysed herein have non-functional TGF-beta pathways. The majority of cells harbor alterations in at least one of the putative components of TGF-beta pathways, mainly in p15 and Smad4 genes. These results suggest that inactivation of TGF-beta signaling pathways plays an important role in human pancreatic tumorigenesis.

The autoantigen La/SSB is a calmodulin-binding protein.

The work reported here has been directed to the identification of new nuclear calmodulin-binding proteins. To achieve this goal, nuclei from rat hepatocytes were purified and a fraction enriched in DNA- and RNA-binding proteins was extracted using DNase I and RNase A. Calmodulin-binding proteins present in this nuclear subfraction were purified by chromatography using first a DEAE-Sephacel column and subsequently a calmodulin-Sepharose column. Four major polypeptides of 118, 107, 48 and 45 kDa were found to bind to the calmodulin column in a Ca(2+)-dependent way. [125I]-calmodulin overlay analysis confirmed that the proteins of 118, 48 and 45 kDa are calmodulin-binding proteins. These proteins bind single-stranded and also double-stranded DNA. A partial amino acid sequence obtained from the 48 kDa protein revealed a 100% identity with the La/SSB protein, an autoantigen implicated in several autoimmune diseases, such as lupus erythematosus and Sjögren's syndrome. Two-dimensional gel electrophoresis, Western blot analysis and experiments of binding to poly(U), also supports the identity of p48 as La/SSB. CaM and La/SSB protein colocalize in the heterochromatinic regions within the nucleus of rat hepatocytes. Preincubation of La/SSB with calmodulin in the presence of Ca2+ resulted in an increase in the binding of ssDNA to La/SSB, suggesting that calmodulin can play a role in the regulation of the association of La/SSB with DNA.

Addition of calmodulin antagonists to NRK cells during G1 inhibits proliferating cell nuclear antigen expression.

The mRNAs of most proteins involved in DNA synthesis show an S phase correlated expression when mammalian cells are stimulated to proliferate from G0. This is the case for proliferating cell nuclear antigen (PCNA), a cofactor of DNA polymerase delta that is essential for the synthesis of the leading and lagging strands of DNA. Normal rat kidney cells re-entering the cell cycle from quiescence start DNA synthesis at 12 h and reach a maximum at 20 h. The expression of PCNA parallels the synthesis of DNA. Progression through the S phase was inhibited by addition of the anticalmodulin drug W13 to the cells during G1, 5 h after activation. W13 also inhibited the increase in both PCNA protein and mRNA indicating that calmodulin regulates its expression. Using TK-ts13 cells transfected with a plasmid containing the thymidine kinase gene under the control of the human 2.8 kb PCNA promoter, we demonstrated that this promoter is not regulated by calmodulin. The half-life of PCNA mRNA during G1/S transition was not modified by the treatment with W13, indicating that the decrease in the mRNA found when calmodulin was inhibited is not due to changes in its stability. Run-on assays revealed that control cells produced predominantly complete PCNA transcripts during S phase, while short incomplete transcripts were generated in W13-treated cells at the same time. These results indicate that calmodulin participates in a more direct or indirect way during G1 in the activation of PCNA expression. From data presented here it can be suggested that calmodulin activates the release of a transcriptional block leading to an increase in the amount of PCNA during S phase.

Calmodulin expression during proliferative activation of human T lymphocytes.

We have investigated the levels of calmodulin mRNA species and calmodulin protein during proliferation of human T lymphocytes. Quiescent lymphocytes expressed the 1.7 kb transcript of CaM I, the 1.4 kb of CaM II and the 2.3 kb of CaM III. Phytohaemagglutinin added to peripheral blood lymphocytes induced DNA replication which started at 48 h and reached a maximum at 72 h after activation. All the species of calmodulin mRNAs, including the 4.0 kb transcript of CaM I and the 0.8 kb of CaM III which were not detected in quiescent cells, increased during lymphocyte proliferation. At 72 h after activation, the increase of CaM I and CaM II transcripts were found to be 2-fold whereas CaM III mRNAs increased 9-fold. The cellular content of calmodulin protein was also found to increase during proliferation and calmodulin accumulations in cytosol and nuclei of activated cells were observed. Two calmodulin binding proteins of 180 and 170 kD were found to increase in the nuclei of proliferating lymphocytes, whereas on the contrary 3 other calmodulin binding proteins of 110, 62 and 60 kD decreased during proliferation.

Protein kinase C regulates calmodulin expression in NRK cells activated to proliferate from quiescence.

We have investigated the levels of calmodulin protein and calmodulin mRNA species during proliferative activation of NRK cells. Cells activated to proliferate from quiescence started to replicate DNA at 15 h, reaching a maximum at 20 h after serum addition. The maximum of mitosis was observed at 24 h. Quiescent cells showed a calmodulin concentration of 1.5 ng/micrograms of protein. At 10 h after serum addition the amount of calmodulin started to increase, reaching values of 3.0 ng/micrograms of protein at 24 h. NRK cells expressed predominantly 3 species of calmodulin transcripts: the 1.7 kb from CaM I, the 1.4 kb from CaM II and the 2.3 kb from CaM III. The amount of all the 3 transcripts was low in quiescent cells and 10 h after activation the levels were already high, reaching a maximum around 20 h. At the latter time the amount of the 3 calmodulin mRNAs was 5-10-fold higher than in serum starved cells. Run-on experiments showed that at 20 h after activation the transcription rates of the 3 calmodulin genes were higher than in quiescent cells. The addition of protein kinase C inhibitors to the cultures blocked the increase of the calmodulin transcripts while inhibitors of protein kinase A did not have any effect. Moreover, the addition of submitogenic doses of phorbol 12-tetradecanoate induced the increase of all 3 calmodulin transcripts. These results indicate that protein kinase C regulates calmodulin expression when NRK cells are activated to proliferate.

New nuclear functions for calmodulin.

The data reported here summarize a series of results which reveal new functions for nuclear calmodulin (CaM). The addition of CaM inhibitors to cultures of proliferating NRK cells blocked the activity of the cyclin-dependent protein kinases 4 (cdk4) and 2 (cdk2), which are enzymes implicated in the progression of G1 and in the onset of DNA replication, respectively. CaM modulates the activity of cdk4 by regulating the nuclear location of both cdk4 and cyclin D, its associated regulatory subunit. By using CaM-affinity chromatography, we have recently identified two new nuclear CaM-binding proteins: (i) the protein La/SSB, which is an autoantigen implicated in several autoimmune diseases such as lupus erythematosus and Sjögren's syndrome (since La/SSB participates in the process of transcription mediated by RNA polymerase III, CaM could be involved in the regulation of this process); and (ii) the protein SAP145, a member of the spliceosome-associated proteins (SAPs) which is a subunit of the splicing factor SF3(b). This finding suggests the involvement of CaM in pre-mRNA splicing. Finally, a screening for new CaM-binding proteins in the fission yeast performed by using the phage display analysis, revealed that several nucleolar-ribosomal proteins associate to CaM, suggesting that CaM modulates ribosomal assembly and/or function.

Changes in nuclear content of protein conjugate histone H2A-ubiquitin during rooster spermatogenesis.

Electrophoretic analysis of acid-soluble chromosomal proteins isolated from rooster testis cell nuclei at different stages of spermatogenesis, revealed that the nuclear content of a protein identified by its solubility, electrophoretic mobility and amino acid analysis as the protein conjugate histone H2A-ubiquitin (uH2A, A24) changed markedly from meiotic cells to late spermatids. The protein was not detectable in tetraploid primary spermatocytes; it was present in 1.7% of the total amount of nucleosomal core histones in early spermatids and reached its maximum level (3.5% and 11%) at the end of spermiogenesis, when histones are replaced by the protamine galline.

Ubiquitin gene expression is increased in skeletal muscle of tumour-bearing rats.

Rats bearing the fast-growing AH-130 Yoshida ascites hepatoma showed a marked cachectic response which has been previously reported [Tessitore et al. (1987) Biochem. J. 241, 153-159]. Thus tumour-bearing animals showed significant decreases in body and muscle weight (soleus and gastrocnemius) as compared to both pair-fed and ad libitum-fed animals. These decreases were related to an enhanced proteolytic rate in the muscles of the tumour-bearing animals as measured by the tyrosine released in in vitro assays. In an attempt to elucidate which proteolytic system is directly responsible for the decrease in muscle mass, we have studied both lysosomal and non-lysosomal (ATP-dependent) proteolytic systems in this animal model. While the enzymatic activities of the main cathepsin (B and B + L) systems were actually decreased in gastrocnemius muscles of tumour-bearing rats, thus indicating that lysosomal proteolysis was not involved, the ubiquitin pools (both free and conjugated) were markedly altered as a result of tumour burden. These were associated with an increased ubiquitin gene expression in muscle of tumour-bearing rats, over 500% in relation to non-tumour bearers, thus suggesting that the ATP-dependent proteolytic system may be responsible for the muscle proteolysis and wastage observed in this animal tumour model. The fact that we have previously shown that TNF enhances the ubiquitinization of muscle proteins [García-Martínez et al. (1993) FEBS Lett. 323, 211-214], together with the high circulating levels of TNF detected in rats bearing the Yoshida hepatoma allows us to suggest that the cytokine may be responsible, most probably indirectly, for the activation of the referred proteolytic system in tumour-bearing rats.

Tumour necrosis factor-alpha increases the ubiquitinization of rat skeletal muscle proteins.

An acute intravenous administration of 100 micrograms/kg body weight of recombinant tumour necrosis factor-alpha (TNF) resulted in a time-dependent increase in the levels of both free and conjugated ubiquitin in rat skeletal muscle. The effects of the cytokine were more pronounced in the red muscle soleus than in the white muscle EDL. In the former muscle type, TNF-treatment also resulted in a time-dependent increase in the percentage of free ubiquitin. The results suggest that the ubiquitin system for non-lysosomal protein degradation could have a very important role in the mechanism triggered by TNF which is responsible for enhanced muscle proteolysis in sepsis and other pathological states.

Protein turnover in skeletal muscle of tumour-bearing transgenic mice overexpressing the soluble TNF receptor-1.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and transgenic mice for the soluble TNF receptor type I protein (sTNF-R1) resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the transgenic mice muscle waste was not affected to the same extent as in the wild-type group. Muscle waste in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result was a decreased rate of protein accumulation which accounted for the muscle weight loss observed as a result of the tumour burden. In contrast, transgenic mice did not have such low rates of protein accumulation after tumour implantation. The increase in protein degradation in the tumour-bearing transgenic mice was accompanied by a similar increase in protein synthesis which compensated for the loss of muscle protein by degradation. Both tumour-bearing groups showed an enhanced expression of ubiquitin and proteasome C8 subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. It is suggested that TNF may, in part, be responsible for the loss of protein in skeletal muscle of tumour-bearing mice.

Muscle hypercatabolism during cancer cachexia is not reversed by the glucocorticoid receptor antagonist RU38486.

In rats into which a fast growing ascites hepatoma (Yoshida AH-130) had been transplanted, tumor growth elicited a marked loss of body weight and tissue waste, particularly of the skeletal muscle. This depletion has been associated with enhanced rates of protein breakdown, mainly due to hyperactivation of the ATP-ubiquitin-dependent proteolytic system [Llovera, M., García-Martínez, C., Agell, N., Marzábal, M., López-Soriano, F.J. and Argilés, J.M. (1994) FEBS Lett., 338, 311-318]. Profound alterations of the hormonal status and the production of tumor necrosis factor have been involved in the development of such wasting syndrome [Tessitore, L., Costelli, P. and Baccino, F.M. (1993) Br. J. Cancer, 67, 15-23]. In the present study, the role of glucocorticoids in muscle hypercatabolism was investigated using the glucocorticoid receptor antagonist RU38486. The treatment with this drug was unable to interfere with the development of cachexia in the AH-130 hosts with regard to tissue weight as well as to muscle protein turnover rates. As one would expect, the RU38486 was also ineffective in lowering both the expression of ubiquitin mRNA and the degree of muscle protein ubiquitinization in AH-130 bearers. These data allow us to exclude that glucocorticoids play a direct crucial role in the development of cachexia in this tumor model.

Different cytokines modulate ubiquitin gene expression in rat skeletal muscle.

Intravenous administration of different cytokines caused important changes in the expression of ubiquitin genes in skeletal muscle. Tumour necrosis factor-alpha caused a 2.2- and 1.9-fold increase in the expression of the 2.4 and 1.2 kb transcripts, respectively. Administration of interferon-gamma also caused a 2.2- and 1.8-fold increase in the 2.4 and 1.2 kb transcripts, respectively. While administration of leukaemia inhibitory factor and interleukin-6 resulted in no changes in ubiquitin gene expression, interleukin-1 administration also caused an increase in both ubiquitin gene transcripts (2.8- and 1.9-fold for the 2.4 and 1.2 kb transcripts, respectively). The results suggest that some of the cytokine effects on the ubiquitin system gene expression could be related to the enhanced skeletal muscle proteolysis found during cancer cachexia and other pathological states.

Role of TNF receptor 1 in protein turnover during cancer cachexia using gene knockout mice.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and gene-deficient mice for the TNF-alpha receptor type I protein (Tnfr1 degree/Tnfr1 degree), resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the gene-knockout mice muscle wastage was not affected to the same extent. In both groups, tumour burden resulted in significant increases in circulating TNF-alpha, a cytokine which, as we have previously demonstrated, can induce protein breakdown in skeletal muscle. Muscle wastage in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result is a decreased rate of protein accumulation that accounts for the muscle weight loss observed as a result of tumour burden. In contrast, gene knockout mice did not have significantly lower rates of protein accumulation as a result of tumour implantation. The increase in protein degradation in the tumour-bearing wild mice was accompanied by an enhanced expression of both ubiquitin and proteasome subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. Tumour-bearing gene-deficient mice did not show any increase in gene expression. It is concluded that TNF-alpha (alone or in combination with other cytokines) is responsible for the activation of protein breakdown in skeletal muscle of tumour-bearing mice.

Ubiquitin gene expression is increased in human muscle undergoing neurogenic involvement.

Histological features of neurogenic muscle involvement included type grouping, muscle fiber atrophy, and target fibers. In muscles with myofiber atrophy and target fibers, we found an increased expression of the genes encoding for the ubiquitin-ATP-dependent proteolytic system. Thus, in patients with target fibers, a 5.2- and a 3.9-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively, while in those with atrophic angulated hyperoxidative fibers, a 3.9- and a 4.4-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively. It is suggested that the activation of this proteolytic system may be responsible for the skeletal muscle alterations that often accompany human muscle neurogenic involvement.