Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process ...of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. oxaliplatin, doxorubicin, mitoxantrone, methotrexate), (ii) early rRNA processing (e.g. camptothecin, flavopiridol, roscovitine), or (iii) late rRNA processing (e.g. 5-fluorouracil, MG-132, homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, whereas blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens.
The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we ...examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17-36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2--simulating the high prevalence of AAV2 antibodies in the population--even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.
High concentrations (> 100 µM) of the ribonucleoside analog 4-thiouridine (4sU) is widely used in methods for RNA analysis like photoactivatable-ribonucleoside-enhanced crosslinking and ...immunoprecipitation (PAR-CLIP) and nascent messenger (m)RNA labeling (4sU-tagging). Here, we show that 4sU-tagging at low concentrations ≤ 10 µM can be used to measure production and processing of ribosomal (r)RNA. However, elevated concentrations of 4sU (> 50 µM), which are usually used for mRNA labeling experiments, inhibit production and processing of 47S rRNA. The inhibition of rRNA synthesis is accompanied by nucleoplasmic translocation of nucleolar nucleophosmin (NPM1), induction of the tumor suppressor p53, and inhibition of proliferation. We conclude that metabolic labeling of RNA by 4sU triggers a nucleolar stress response, which might influence the interpretation of results. Therefore, functional ribosome biogenesis, nucleolar integrity, and cell cycle should be addressed in 4sU labeling experiments.
CD73 catalyzes the conversion of ATP to adenosine, which is involved in various physiological and pathological processes, including tumor immune escape. Because CD73 expression and activity are ...particularly high on cancer cells and contribute to the immunosuppressive properties of the tumor environment, it is considered an attractive target molecule for specific cancer therapies. In line, several studies demonstrated that CD73 inhibition has a significant antitumor effect. However, complete blocking of CD73 activity can evoke autoimmune phenomena and adverse side effects. We developed a CD73-specific antibody, 22E6, that specifically inhibits the enzymatic activity of membrane-tethered CD73 present in high concentrations on cancer cells and cancer cell-derived extracellular vesicles but has no inhibitory effect on soluble CD73. Inhibition of CD73 on tumor cells with 22E6 resulted in multiple effects on tumor cells in vitro, including increased apoptosis and interference with chemoresistance. Intriguingly, in a xenograft mouse model of acute lymphocytic leukemia (ALL), 22E6 treatment resulted in an initial tumor growth delay in some animals, followed by a complete loss of CD73 expression on ALL cells in all 22E6 treated animals, indicating tumor immune escape. Taken together, 22E6 shows great potential for cancer therapy, favorably in combination with other drugs.
6A10 is a CA XII inhibitory monoclonal antibody, which was demonstrated to reduce the growth of cancer cells in vitro and in a xenograft model of lung cancer. It was also shown to enhance ...chemosensitivity of multiresistant cancer cell lines and to significantly reduce the number of lung metastases in combination with doxorubicin in mice carrying human triple-negative breast cancer xenografts. Starting from these data, we report here on the development of the 6A10 antigen-binding fragment (Fab), termed Fab6A10, and its functional, biochemical, and structural characterization. In vitro binding and inhibition assays demonstrated that Fab6A10 selectively binds and inhibits CA XII, whereas immunohistochemistry experiments highlighted its capability to stain malignant glioma cells in contrast to the surrounding brain tissue. Finally, the crystallographic structure of CA XII/Fab6A10 complex provided insights into the inhibition mechanism of Fab6A10, showing that upon binding, it obstructs the substrate access to the enzyme active site and interacts with CA XII His64 freezing it in its out conformation. Altogether, these data indicate Fab6A10 as a new promising therapeutic tool against cancer.
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•Inhibition of CA XII in tumors expressing this protein has a great antineoplastic potential.•6A10 is a CA XII inhibitory monoclonal antibody.•In this paper, we report on the development and characterization of a 6A10 Fab fragment.•Our findings indicate Fab6A10 as a promising therapeutic tool against cancer.
PeBoW, a trimeric complex consisting of pescadillo (Pes1), block of proliferation (Bop1), and the WD repeat protein 12 (WDR12), is essential for processing and maturation of mammalian 5.8S and 28S ...ribosomal RNAs. Applying a mass spectrometric analysis, we identified the DEAD-box helicase DDX27 as stably associated factor of the PeBoW-complex. DDX27 interacts with the PeBoW-complex via an evolutionary conserved F×F motif in the N-terminal domain and is recruited to the nucleolus via its basic C-terminal domain. This recruitment is RNA-dependent and occurs independently of the PeBoW-complex. Interestingly, knockdown of DDX27, but not of Pes1, induces the accumulation of an extended form of the primary 47S rRNA. We conclude that DDX27 can interact specifically with the Pes1 and Bop1 but fulfils critical function(s) for proper 3′ end formation of 47S rRNA independently of the PeBoW-complex.
•DEAD-box helicase DDX27 is a new constituent of the PeBoW-complex.•The N-terminal F×F motif of DDX27 interacts with the PeBoW components Pes1 and Bop1.•Nucleolar anchoring of DDX27 via its basic C-terminal domain is RNA dependent.•Knockdown of DDX27 induces a specific defect in 3′ end formation of 47S rRNA.
Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent ...kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.
Background: Processing of ribosomal RNA is sensitive to Cdk inhibitors.
Results: Inhibition of Cdk9 activity blocks 47 S rRNA processing and stabilizes a 3′ extended 47 S primary transcript. Defective 3′ processing negatively feeds back on RNAPI transcription.
Conclusion: Cdk9 facilitates processing of 47 S rRNA by RNAPII-dependent synthesis of U8 snoRNA.
Significance: Cdk9 may be a critical regulator of rRNA processing to harmonize RNAPII transcription activity with a ribosome biogenesis rate.
Since its first description more than 30 years ago p53 has become a paradigm for a protein with versatile functions. P53 sensitizes a large variety of genetic alterations and has been entitled the ...guardian of the genome. Stabilization of p53 upon DNA damage is accompanied by a complex pattern of modifications, which ascertain the cellular response either in the direction of a reversible or irreversible cell cycle arrest or programmed cell death. More recently it became evident that p53 also responds to non-genotoxic cell stress, in particular if ribosome biogenesis is affected.
Early aldosterone up-regulated genes: New pathways for renal disease?
The use of aldosterone antagonists has important beneficial effects on the progression of renal and cardiac disease reflected in ...a decrease of cardiovascular mortality and renal failure. Nevertheless, the incidence of heart and end-stage renal failure continues to increase. This leads to the conclusion that mechanisms independent of the classical aldosterone/mineralocorticoid receptor system may contribute to the pathogenesis of cardiac and renal disease.
The mRNA expression profile of human renal epithelial cells in response to aldosterone was characterized using cDNA arrays covering ∼1430 genes. Differentially expressed genes were further evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and estimating the gene products by Western blotting.
Aldosterone treatment of cells resulted in significant up-regulation of several genes within 1 hour, with sgk, p21/waf1, gadd45, and gadd153 being the most significant ones. Long-term treatment (>4 hours) with aldosterone induced the mRNA expression of pparα and purα. The mineralocorticoid receptor inhibitor spironolactone decreased the mRNA levels of sgk, p21/waf1, and gadd45, whereas the glucocorticoid receptor inhibitor RU 486 reduced the mRNA level of sgk and p21/waf1. Gadd153 was not affected by any of the inhibitors, probably indicating regulation by nonclassic mechanisms.
Among the early genes investigated in this study, one transcript has been identified that is not suppressed by antagonists of either glucocorticoid or mineralocorticoid receptor. Further studies should be able to identify other genes regulated in a similar manner that could explain the inefficacy of spironolactone in some cases of aldosterone-mediated kidney disease.
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