Sebastian Patzke

Identification of a novel centrosome/microtubule-associated coiled-coil protein involved in cell-cycle progression and spindle organization

Here we describe the identification of a novel vertebrate-specific centrosome/spindle pole-associated protein (CSPP) involved in cell-cycle regulation. The protein is predicted to have a tripartite domain structure, where the N- and C-terminal domains are linked through a coiled-coil mid-domain. Experimental analysis of the identified domains revealed that spindle association is dependent on the N-terminal and the coiled-coil mid domain. The expression of CSPP at the mRNA level was detected in all tested cell lines and in testis tissue. Ectopic expression of CSPP in HEK293T cells blocked cell-cycle progression in early G1 phase and in mitosis in a dose-dependent manner. Interestingly, mitosis-arrested cells contained aberrant spindles and showed impairment of chromosome congression. Inhibition of CSPP gene expression by small interfering RNAs induced cell-cycle arrest/delay in S phase. This phenotype was characterized by elevated levels of cyclin A, decreased levels of cyclin E and hyperphosphorylation of the S-phase checkpoint kinase Chk1. The activation of Chk1 may indicate a replication stress response due to an inappropriate G1/S-phase transition. Taken together, we demonstrate that CSPP is associated with centrosomes and microtubules and may play a role in the regulation of G1/S-phase progression and spindle assembly.

CSPP Is a Ciliary Protein Interacting with Nephrocystin 8 and Required for Cilia Formation

CSPP and CSPP-L are centrosomal proteins of known mitotic function. Here, we identify CSPP proteins as ciliary proteins and place them into a NPHP protein network crucial for normal cilia-dependent renal and retinal tissue architecture. Importantly, CSPP-L is found to be required for ciliogenesis and shown to be a cilia length modulator.

Three independent mechanisms for arrest in G2after ionizing radiation

Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G2-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G2-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G2 phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2hours after IR (0.5 or 4Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6hours after IR) of cells that were in G2/S/G1 at the time of irradiation (4Gy) was inactivated by caffeine. This late G2-arrest could not be explained by down-regulation of genes with functions in G2/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1–depleted cells. TP53 was not necessary for the late G2-arrest, but mediated an intermediate arrest (2-10hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G2 is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.

CSPP and CSPP-L associate with centrosomes and microtubules and differently affect microtubule organization.

We recently described the identification of a centrosome/spindle pole associated protein, CSPP, involved in cell cycle progression. Here we report a CSPP isoform denoted CSPP‐L, with a 294 amino acids longer N‐terminus and a 51 amino acids insertion located in the coiled‐coil mid‐domain. Expression analysis indicates an inverse cell cycle dependent regulation. CSPP mRNA expression is highest in G1 whereas CSPP‐L expression is highest in G2/M. Ectopic expression of CSPP‐L impairs cell cycle progression weaker in G1 than CSPP. Furthermore, normal mitotic phenotypes were observed in CSPP‐L but not in CSPP transfectants. CSPP‐L relocates from spindle microtubules and poles in metaphase to the mid‐spindle in anaphase and concentrates at the mid‐body in telophase/cytokinesis. CSPP‐L high‐expressing mitotic cells were predominantly characterized by lagging chromosomes or monopolar spindles, in contrast to the predominant multipolar spindles observed with CSPP expression. The different effects of CSPP and CSPP‐L on microtubule organization in mitosis depend on the coiled‐coil mid‐domain insertion. The common C‐terminal domain is required to repress that activity until mitosis. Notably, this C‐terminal domain alone can associate with centrosomes in a microtubule independent manner. Taken together, CSPP and CSPP‐L interact with centrosomes and microtubules and can differently affect microtubule organization. J. Cell. Physiol. 209: 199–210, 2006.

Replication-induced DNA damage after PARP inhibition causes G2 delay, and cell line-dependent apoptosis, necrosis and multinucleation

PARP inhibitors have been approved for treatment of tumors with mutations in or loss of BRCA1/2. The molecular mechanisms and particularly the cellular phenotypes resulting in synthetic lethality are not well understood and varying clinical responses have been observed. We have investigated the dose- and time-dependency of cell growth, cell death and cell cycle traverse of 4 malignant lymphocyte cell lines treated with the PARP inhibitor Olaparib. PARP inhibition induced a severe growth inhibition in this cell line panel and increased the levels of phosphorylated H2AX-associated DNA damage in S phase. Repair of the remaining replication related damage caused a G2 phase delay before entry into mitosis. The G2 delay, and the growth inhibition, was more pronounced in the absence of functional ATM. Further, Olaparib treated Reh and Granta-519 cells died by apoptosis, while U698 and JVM-2 cells proceeded through mitosis with aberrant chromosomes, skipped cytokinesis, and eventually died by necrosis. The TP53-deficient U698 cells went through several rounds of DNA replication and mitosis without cytokinesis, ending up as multinucleated cells with DNA contents of up to 16c before dying. In summary, we report here for the first time cell cycle-resolved DNA damage induction, and cell line-dependent differences in the mode of cell death caused by PARP inhibition.

Targeted cancer therapy with a novel anti-CD37 beta-particle emitting radioimmunoconjugate for treatment of non-hodgkin lymphoma

177Lu-DOTA-HH1 (177Lu-HH1) is a novel anti-CD37 radioimmunoconjugate developed to treat non-Hodgkin lymphoma. Mice with subcutaneous Ramos xenografts were treated with different activities of 177Lu-HH1, 177Lu-DOTA-rituximab (177Lu-rituximab) and non-specific 177Lu-DOTA-IgG1 (177Lu-IgG1) and therapeutic effect and toxicity of the treatment were monitored. Significant tumor growth delay and increased survival of mice were observed in mice treated with 530 MBq/kg 177Lu-HH1 as compared with mice treated with similar activities of 177Lu-rituximab or non-specific 177Lu-IgG1, 0.9% NaCl or unlabeled HH1. All mice injected with 530 MBq/kg of 177Lu-HH1 tolerated the treatment well. In contrast, 6 out of 10 mice treated with 530 MBq/kg 177Lu-rituximab experienced severe radiation toxicity. The retention of 177Lu-rituximab in organs of the mononuclear phagocyte system was longer than for 177Lu-HH1, which explains the higher toxicity observed in mice treated with 177Lu-rituximab. In vitro internalization studies showed that 177Lu-HH1 internalizes faster and to a higher extent than 177Lu-rituximab which might be the reason for the better therapeutic effect of 177Lu-HH1.

CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation

Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.

New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed “G2H” compartment), to cells that there were almost negative and had about 2 foci (termed “G2L” compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2–3 h delay in G2L.

The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia formation via ubiquitylation of CSPP-L.

Primary cilia are crucial for signal transduction in a variety of pathways, including Hedgehog and Wnt. Disruption of primary cilia formation (ciliogenesis) is linked to numerous developmental disorders (known as ciliopathies) and diseases, including cancer. The Ubiquitin-Proteasome System (UPS) component UBR5 was previously identified as a putative modulator of ciliogenesis in a functional genomics screen. UBR5 is an E3 Ubiquitin ligase that is frequently deregulated in tumours, but its biological role in cancer is largely uncharacterised, partly due to a lack of understanding of interacting proteins and pathways. We validated the effect of UBR5 depletion on primary cilia formation using a robust model of ciliogenesis, and identified CSPP1, a centrosomal and ciliary protein required for cilia formation, as a UBR5-interacting protein. We show that UBR5 ubiquitylates CSPP1, and that UBR5 is required for cytoplasmic organization of CSPP1-comprising centriolar satellites in centrosomal periphery. Hence, we have established a key role for UBR5 in ciliogenesis that may have important implications in understanding cancer pathophysiology.