Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed

The phototoxic red fluorescent GFP-like protein KillerRed has recently been described. The phototoxicity of KillerRed exceeds that of EGFP by at least 1,000-fold, making it the first fully genetically encoded photosensitizer. KillerRed opens up new possibilities for precise light-induced cell killing and target protein inactivation. Because KillerRed is encoded by a gene, it can be expressed in a spatially and temporally regulated manner, under a chosen promoter, and fused with the desired protein of interest or localization signal. Here we provide a protocol for target protein inactivation in cell culture using KillerRed. As KillerRed is a new tool, the protocol focuses on aspects that will allow users to maximize the potential of this protein, guiding the design of chimeric constructs, recommended control experiments and preferred illumination parameters. The protocol, which describes target protein visualization and subsequent inactivation, is a 2- or 3-d procedure.     

Heavy ion effects on yeast: inhibition of ribosomal RNA synthesis

Diploid wild-type yeast cells were exposed to beams of heavy ions covering a wide range of linear energy transfer (LET) (43-13,700 keV/microns). Synthesis of ribosomal RNA (rRNA) was assessed as a functional measure of damage produced by particle radiation. An exponential decrease of relative rRNA synthesis with particle fluence was demonstrated in all cases. The inactivation cross sections derived were found to increase with LET over the entire range of LET studied. The corresponding values for relative biological effectiveness were slightly less than unity. Maximum cross sections measured were close to 1 micron 2, implying that some larger structure within the yeast nucleus (e.g., the nucleolus) might represent the target for an impairment of synthetic activity by very heavy ions rather than the genes coding for rRNA. Where tested, an oxygen effect for rRNA synthesis could not be demonstrated.     

Correlated inhibition of ribosomal RNA synthesis and silver staining by actinomycin D

Summary Preferential inhibition of the synthesis of ribosomal RNA by low doses of actinomycin D was used to investigate the quantitative relationship between the intensity of silver staining of nucleoli and the rate of RNA-synthesis. The two parameters were found to be strongly correlated in human diploid fibroblasts.     

Chromophore-assisted laser inactivation (CALI) to elucidate cellular mechanisms of cancer.

Chromophore-assisted laser inactivation (CALI) is a new technology for acute protein inactivation in living cells. It targets laser energy to specific proteins via non-function-blocking antibodies that are labeled with the dye malachite green. Excitation of the dye generates short-lived free radicals that damage the bound protein without affecting other cellular components. The wavelength of laser light used (620 nm) is not readily absorbed by cells such that non-specific light damage does not occur. CALI provides an alternative to other inactivation strategies and has the advantages of high spatial and temporal resolution. The ultimate value of this technology for cancer research will be assessed by how effective CALI is in ascribing in situ function during cancer-relevant processes and in identifying and validating protein targets for drug discovery. Recent work using CALI on ezrin and pp60-c-src, two proteins that may be involved in cancer, suggests its potential. Further application of CALI will likely be of utility for understanding cellular mechanisms of cancer and developing cancer therapeutics.     

Reversible inhibition of ribosomal RNA synthesis in HeLa by lucanthone (Miracil D) with continued synthesis of DNA-like RNA

Ribosomal RNA synthesis was selectively inhibited in HeLa cells by lucanthone, a clinically useful schistosomicide which shares many of the properties of Actinomycin D. Synthesis of DNA-like RNA continued during complete inhibition of ribosomal RNA synthesis. Under these conditions newly synthesized DNA-like RNA accumulated normally in polyribosomes of the cell cytoplasm; most of it appeared to be messenger RNA. DNA synthesis was partially inhibited by lucanthone but protein synthesis was undisturbed. Synthesis of ribosomal RNA promptly resumed after removal of lucanthone and cell survival was not affected if exposures to the drug were limited to two hours.     

Bacteriophage P2-lambda interference: inhibition of protein synthesis involves transfer RNA inactivation

Upon induction of bacteriophage λ in an Escherichia coli strain dilysogenic for phage P2 and λ, incorporation of all radioactive precursors is severely depressed. It appears, however, that incorporation into proteins is shut off before incorporation into RNA. Experiments with acellular extracts showed that all components of the translation machinery remain active in vitro, except the tRNA fraction. tRNA presents a functional alteration which affects its aminoacylation as well as the transfer of the activated complex on ribosomic sites. I thank Professor Francois Gros for guidance and counsel, and for providing laboratory facilities, Dr Philippe Kourilsky for constructing the dilysogenic strain and for stimulating discussions all through the course of this work and Dr Moshe Yaniv for useful advice about tRNA processing.     

Selective inhibition of ribosomal RNA synthesis by rifampicin in E. coli cells]

Under partial inhibition of total RNA synthesis by rifampicin the formation of beta- and beta'-subunits of RNA polymerase is stimulated and the rRNA synthesis is selectively repressed. The differential rate of synthesis of the beta- and beta'-subunits increases from 1,15% up to 2,88% in the presence of 30 micrograms rifampicin per ml. Simultaneously the differential rate of rRNA synthesis decreases from 41% down to 10%. The degree of inhibition of rRNA synthesis by rifampicin depends on the cell growth rate.     

Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells

To investigate the effect of small-interfering RNA (siRNA) targeted against Ki-67, which is an attractive molecular target for cancer therapy, on inhibiting Ki-67 expression and cell proliferation in human renal carcinoma cells (HRCCs), siRNAs were used to inhibit the expression of Ki-67 in HRCCs. Ki-67 mRNA levels were detected by RT-PCR and in situ hybridization analysis. Ki-67 protein levels were detected by Western blot and immunocytochemistry analysis. TUNEL assay was used to measure the apoptosis of carcinoma cells. Results of RT-PCR and in situ hybridization demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells. Similar reduction in Ki-67 protein measured by Western blot and immunocytochemistry was observed in cells transfected with Ki-67 siRNA. Ki-67-siRNA treatment of HRCCs resulted in specific inhibition of proliferation and increased apoptotic cell death. From these findings we conclude that inhibition of Ki-67 expression by siRNA may be a reasonable approach in renal cancer therapy.     

Comparison of the action of light on ribosomal RNA synthesis in cabbage and mustard seedlings

We have compared the action of light on ribosomal RNA synthesis in mustard and cabbage seedlings, two of the most frequently used systems for the studies of anthocyanin synthesis. The level of RNA (both t-RNA and r-RNA) “stored” in mustard dry seeds is much lower than in cabbage dry seeds. The kinetics of RNA synthesis in cabbage and mustard seedlings exposed to light are very different: In cabbage seedlings, light produces no apparent stimulation of cytoplasmic r-RNA synthesis, while it does increase plastid r-RNA synthesis. On the other hand, in mustard seedlings, light promotes both cytoplasmic and plastid ribosomal RNA synthesis. Streptomycin, which inhibits chlorophyll formation and chloroplast development while having no effect (mustard) or enhancing (cabbage) anthocyanin synthesis in these two systems, is in both cases an effective inhibitor of plastid r-RNA synthesis, but not of cytoplasmic r-RNA synthesis.     

Effect of blue and red light on the synthesis of ribosomal RNA in Chlorella]

In autotrophic cultures of Chlorella pyrenoidosa (strain 211-8b) incorporation of tritiated guanosine and uridine into ribosomal RNA is stimulated by light. Blue light of wavelengths around 457 nm is considerably more effective than red light around 679 nm (5-10(-10) Einstein cm-2 sec-1 for both). This effect can be demonstrated for young daughter cells (at the end of the dark period) and for older cells (at the end of the light period). It is shown to depend on a regulation of rRNA-synthesis. The blue light dependent enhancement of incorporation is more pronounced in the cytoplasmic rRNA (25 and 18 s) than in the chloroplast rRNA (23 and 16 s). Blue light of low intensity (1-10(-10) Einstein cm-2 sec-1) has nearly the same effectivity as the fivefold intensity, whereas red light of equal quantum fluxes enhances incorporation only slightly compared with the dark control. The blue light dependent enhancement of rRNA-synthesis continues in the following darkness in contrary to that caused by red light. This enhancement is also found in DCMU-poisened cultures. In contrast to this, is red light in presence of DCMU, incorporation into rRNA is nearly the same as in dark. It is concluded that the regulation of rRNA-synthesis in red light is closely connected to complete photosynthesis, while in blue light an additional regulation takes place independent of photosynthesis.