Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Galand, Paul; Degraef, Chantal

doi:10.1111/j.1365-2184.1989.tb00223.x

Cited by 164 publications

(106 citation statements)

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“…This is probably due to the fact that the immunoreactivity of PCNA is affected by factors such as a type of antibodies and of fixatives, a fixation condition and a staining procedure against PCNA. For example, there is a report that whereas monoclonal antibody 19A2 exclusively recognize S-phase cells in methanol-fixed tissues, it reacts with cycling cells in formaldehyde-fixed tissues (10). In contrast, Landberg and Roos (17) did not find such staining characteristics for 19A2 in samples fixed in methanol.…”

Section: Discussionmentioning

confidence: 96%

“…At least two different populations of PCNA have been identified (3,10,20,22,24). It was reported that methanol fixation extracted free PCNA, and that in 3T3 cells, 20-30% of the total PCNA remained even after methanol fixation (3).…”

Section: Discussionmentioning

confidence: 99%

“…However, a great difference in immunoreactivity has been indicated between monoclonal antibodies and autoantibodies (17). The stainability of PCNA is dependent upon the clone of antibodies and is also affected by fixative and/or fixation time (9,10,16,17,22). Even with the same clone of a monoclonal antibody, some controversy still exists concerning the relationship between the expression of PCNA and the cell cycle (13,18).…”

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confidence: 99%

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Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it

1993

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Flow cytometric bivariate DNA/PCNA analysis was performed to investigate the expression of PCNA during the cell cycle and the implication in DNA replication in HeLa cells, using a monoclonal antibody (PC10) to PCNA. The expression of PCNA was evident in almost all cells growing exponentially, when cells were fixed in methanol. The total amount of PCNA altered a little during the cell cycle. However, the treatment with Triton X‐100 extracted 80–89% of total PCNA from the cells, resulting in the dramatic change of bivariate DNA/PCNA distribution pattern. PCNA was completely removed from nuclei in both G1 and G2 phases by the detergent treatment, whereas a certain amount of PCNA remained in S phase nuclei. After the treatment of cells with Triton X‐100, PCNA was detected exclusively in S phase cells. The bivariate DNA/PCNA distribution pattern in cells treated with Triton X‐100 was strikingly so similar to the DNA/BrdUrd distribution pattern that it was unable to differentiate one from the other. It is concluded that the detergent treatment of cells allows the rapid analysis of the cell cycle.The inhibition of DNA synthesis with 10 mM hydroxyurea elevated cellular PCNA content mainly due to the increase in the fraction of the detergent extractable PCNA. It was apparent, however, that in cells incubated with Triton X‐100, the pattern of the bivariate DNA/PCNA distribution was not basically different from that in cells without HU treatment. The level of PCNA bound to nuclear structures (PCNA not extracted with detergent) increased in cells arrested at the G1/S boundary with the time of hydroxyurea treatment. Eventually, it became difficult to differentiate between cells in the G1/S boundary and the early S phase on the basis of DNA and PCNA contents. However, the increase in bound PCNA was minimal in S phase cells. The observation suggests that PCNA is bound to the potential initiation sites of DNA replication together with DNA replication associated enzymes in advance of the commencement of DNA replication and that it is never associated with late replicating DNA without the duplication of early replicating DNA. © 1993 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it

1993

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“…PCNA expression is therefore correlated with the cell cycle. Several authors have suggested this staining as an alternative for thymidine pulse labelling, especially in pre-existing fixed or frozen materials (Galand et al, 1989;Kawakita et al, 1992). We are aware that PCNA staining gives an overestimation of proliferation when compared with 'classical' thymidine incorporation because all cells, except those in Go state, are stained.…”

Section: Discussionmentioning

confidence: 99%

Organ involvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells in the C57BL/KaLwRij mouse

Vanderkerken¹,

Raeve²,

Goes³

et al. 1997

Br J Cancer

133

142

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Summary The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease.Keywords: multiple myeloma; adhesion molecules; organ involvement; 5T2; 5T33 Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal expansion of malignant plasma cells secreting a monoclonal immunoglobulin (Ig). The disease is mainly localized in the bone marrow. In this microenvironment the myeloma plasma cells receive signals necessary for their proliferation, terminal differentiation and for the secretion of osteoclast-activating factors. The osteoclast-activating factors recruit osteoclasts, which induce in situ osteolytic bone lesions (Bataille et al, 1989;Alsina et al, 1996); this is one of the major characteristics of the disease. It has been suggested that both cytokines and adhesion molecules are involved in this complex network of signals (Van Riet and Van Camp, 1993).To elucidate the exact mechanisms described above, an in vivo MM model is necessary. Radl et al (1979) found that 0.5% of ageing C57BL/KaLwRij mice spontanously developed a disease reminiscent of MM. The MM cells isolated from the bone marrow of different mice (5T MM) did not grow in vitro but could be transplanted by intravenous injection into young recipients of the same strain. This transplantable model resembles the human disease in several aspects (Radl et al, 1988): myeloma occurred spontaneously, the frequency of development of the disease is age related, tumour load can be assessed by paraproteinaemia and the (Radl et al, 1988).In order to understand the homing mechanisms of the 5T MM cells to the bone marrow, it was essential to determine accurately the...

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“…In the P3 forebrain, a significant level of m-Msi-1 expression was observed in the tightly packed round cells in the SVZ, which was composed of PCNApositive proliferating cells (Fig. 1 D,E) (Galand and Degraef, 1989), near the anterior part of the lateral ventricle (SVZa), and was also detected in the migratory pathway toward the olfactory bulb (Fig. 1 B).…”

Section: M-msi-1 Expression In the Svz Of The Forebrainmentioning

confidence: 99%

Expression of Neural RNA-Binding Proteins in the Postnatal CNS: Implications of Their Roles in Neuronal and Glial Cell Development

1997

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There is an increasing interest in the role of RNA-binding proteins during neural development. Mouse-Musashi-1 (m-Msi-1) is a mouse neural RNA-binding protein with sequence similarity to Drosophila musashi (d-msi), which is essential for neural development. m-Msi-1 is highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic CNS development. The present study characterized m-Msi-1-expressing cells in the postnatal and adult CNS. Postnatally, m-Msi-1 was expressed in proliferative neuronal precursors in the external granule cell layer of the cerebellum and in the anterior corner of the subventricular zone of the lateral ventricles. In gliogenesis, the persistent expression of m-Msi-1 was observed in cells of the astrocyte lineage ranging from proliferative glial precursors in the subventricular zone (SVZ) to differentiated astrocytes in the parenchyma. In addition, we showed that m-Msi-1 was still expressed in proliferating cells in the adult SVZ, which may contain neural precursor or stem cells. Another neural RNA-binding protein Hu (the mammalian homolog of a Drosophila neuronal RNAbinding protein Elav) was present in postmitotic neurons throughout the development of the CNS, and its pattern of expression was compared with that of m-Msi-1. These observations imply that these two RNA-binding proteins may be involved in the development of neurons and glia by regulating gene expression at the post-transcriptional level. Key words: RNA-binding proteins; postnatal CNS; mouseMusashi-1 (m-Msi-1); Hu; Drosophila musashi; neuronal and glial precursor cells; astrocyte lineageA number of transcription factors that f unction in the proliferation and differentiation of neural precursor cells have been identified. However, the recent discovery of neural-specific RNAbinding proteins raises the possibility that the development of neural cells from the precursors may also be regulated at the post-transcriptional level. We believe that at least two gene families are represented by the neural RNA-binding proteins found in both invertebrates and vertebrates (Okano, 1995;Sakakibara et al., 1996). One, the Elav family, includes Drosophila elav genes (Yao et al., 1993) and their mammalian homologs, the Hu genes (Szabo et al., 1991). Members of this family share extensive sequence similarity with one another, and they seem to be expressed mainly in postmitotic neurons (Okano and Darnell, 1997). The other Msi family is composed of Drosophila musashi (Nakamura et al., 1994), Xenopus laevis nrp-1 (Richter et al., 1990), and their mouse homolog mouse-musashi-1 (m-msi-1) (Sakakibara et al., 1996). d-Msi is required for the two successive asymmetric cell divisions of sensory organ precursors (Nakamura et al., 1994). In contrast to the Elav family, the members of the Msi family are expressed in the neural precursor cells at least during embryonic C NS development (Sakakibara et al., 1996). Although the molecular f unctions and the in vivo RNA targets of Msi and Elav families remain largel...

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Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Cited by 164 publications

References 19 publications

Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it

Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it

Organ involvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells in the C57BL/KaLwRij mouse

Expression of Neural RNA-Binding Proteins in the Postnatal CNS: Implications of Their Roles in Neuronal and Glial Cell Development

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