Chromophore-assisted light inactivation of pKi-67 leads to inhibition of ribosomal RNA synthesis

Abstract

Objectives: Expression of the nuclear Ki-67 protein (pKi-67) is strongly associated with cell proliferation. For this reason, antibodies against this protein are widely used as prognostic tools for the assessment of cell proliferation in biopsies from cancer patients. Despite this broad application in histopathology, functional evidence for the physiological role of pKi-67 is still missing. Recently, we proposed a function of pKi-67 in the early steps of ribosomal RNA (rRNA) synthesis. Here, we have examined the involvement of pKi-67 in this process by photochemical inhibition using chromophore-assisted light inactivation (CALI).

Materials and methods: Anti-pKi-67 antibodies were labelled with the fluorochrome fluorescein 5(6)-isothiocyanate and were irradiated after binding to their target protein.

Results: Performing CALI in vitro on cell lysates led to specific cross-linking of pKi-67. Moreover, the upstream binding factor (UBF) necessary for rRNA transcription was also partly subjected to cross-link formation, indicating a close spatial proximity of UBF and pKi-67. CALI in living cells, using micro-injected antibody, caused a striking relocalization of UBF from foci within the nucleoli to spots located at the nucleolar rim or within the nucleoplasm. pKi-67-CALI resulted in dramatic inhibition of RNA polymerase I-dependent nucleolar rRNA synthesis, whereas RNA polymerase II-dependent nucleoplasmic RNA synthesis remained almost unaltered.

Conclusions: Our data presented here argue for a crucial role of pKi-67 in RNA polymerase I-dependent nucleolar rRNA synthesis.

Figure 1

CALI in HEp‐2 cell lysates. Samples were incubated as indicated. Immunoblot in (a) shows pKi‐67 detected with the MIB‐1 antibody. In non‐irradiated samples (lanes 1–3) the pKi‐67 main bands are visible at approximately 315 and 350 kDa. After irradiation with a 488 nm cw argon laser at 500 mW/cm² for 15 min these bands are absent and a reactivity of higher molecular weight appears (lanes 4–5). In samples from the same experiment Mcm3 (b) and UBF (c) were detected. The main bands of Mcm3 and UBF are clearly visible after CALI against pKi‐67 (compare lanes 4–5 in a with b, c). Double arrows show the characteristic pKi‐67 and the UBF double band. Numbers on left hand side indicate the size in kDa.

Figure 2

Inhibition of nucleolar RNA synthesis by CALI. Cells were injected with antibody conjugates as indicated and analysed for ongoing RNA synthesis as described in Figure 3. The fractions of cells with inhibition of nucleolar RNA synthesis as well as complete inhibition of RNA synthesis were determined by counting 30–60 microinjected cells. Radiant exposure was 570 mW/cm².

Figure 3

Inhibition of nucleolar RNA transcription and protein relocalisation induced by CALI in living cells. Cells were either microinjected with TuBB‐9‐FITC (a–l) or MIB1‐FITC (m–r). In non‐irradiated control cells (a–c) the microinjected TuBB‐9‐FITC antibody (a) accumulates in small foci within the nucleoli (one nucleolus outlined in red) co‐localizing with UBF (b). In contrast, irradiation of cells with a fluorescence microscope for 90 s at 570 mW/cm² (d–l) leads to a prominent redistribution of the microinjected antibody to distinct intranuclear spots (d, g, j). These spots are frequently located at the rim of the nucleoli (arrows), often forming large complexes (j) as well as to smaller spots within the nucleoplasm (arrowheads). Interestingly, these spots completely co‐localize with UBF staining (e, h, k) which is also apparent by the yellow colour in the merged images (i, l) where TuBB‐9 staining is shown in green and UBF staining in red. While control cells exhibit a distinctive RNA synthesis within nucleoli and nucleoplasm (c), irradiation of microinjected cells leads to a dramatic inhibition of the nucleolar rRNA synthesis whereas the nucleoplasmic RNA synthesis is almost unaffected (f). The microinjected MIB‐1‐FITC antibody accumulates at the outer rim of the nucleoli (m) in non‐irradiated control cells (m–o, one nucleolus outlined in red). In contrast to cells microinjected with TuBB‐9‐FITC, after irradiation (p–r) no effect on protein localisation or RNA synthesis could be observed. Bars: 10 µm; g–l: 5 µm.

Figure 4

CALI with TuBB‐9‐FITC does not alter fibrillarin localisation inside the nucleoli. CALI after nuclear microinjection of TuBB‐9‐FITC (right hand cell) does not affect fibrillarin localisation (b) whereas nucleolar RNA synthesis (c) is inhibited (thick arrows). Microinjection into the cytoplasm which is shown as a control, does affect neither fibrillarin localisation nor RNA synthesis (left hand cell, thin arrows). Irradiation 15 min at 570 mJ/cm². Bars 10 µm.