Expression patterns of cyclin D1 and related proteins regulating G1-S phase transition in uveal melanoma and retinoblastoma

Abstract

Background/aims: A checkpoint mechanism in late G1, whose regulation via loss of retinoblastoma protein (pRB) or p16, or overexpression of cyclin D1 or cyclin dependent kinase 4 (CDK4), has been proposed to constitute a common pathway to malignancy. The aims of this study were (a) to compare markers of cell cycle G1-S phase transition in an intraocular tumour with known pRB deficiency (retinoblastoma) and compare it with one with an apparently functional pRB (uveal melanoma); (b) to determine if one of these markers may have a role in the pathogenesis of uveal melanoma; and (c) to determine if there is a difference in cell cycle marker expression following treatment of uveal melanoma and retinoblastoma.

Methods: 90 eyes were enucleated from 89 patients for retinoblastoma (n = 24) or for choroidal or ciliary body melanoma (n = 66). Conventional paraffin sections were assessed for cell type and degree of differentiation. Additional slides were investigated applying standard immunohistochemical methods with antibodies specific for cyclin D1 protein, pRB, p53, p21, p16, BCL-2, and MIB-1.

Results: Cyclin D1 protein and pRB were negative in retinoblastoma using the applied antibodies. In contrast, cyclin D1 protein expression was observed in 65% of uveal melanomas; a positive correlation between cyclin D1 cell positivity and tumour cell type, location, growth fraction, as well as with pRB positivity was observed. p53, p21, and p16 could be demonstrated in both tumours. An inverse relation between p53 and p21 expression was demonstrated in most choroidal melanomas and in some retinoblastomas. Apart from a decrease in the growth fractions of the tumours as determined by MIB-1, a significant difference in the expression of G1-S phase transition markers in vital areas of uveal melanoma and retinoblastoma following treatment with radiotherapy and/or chemotherapy was not observed.

Conclusion: Retinoblastomas and uveal melanomas, two tumours of differing pRB status, differ also in their immunohistochemical pattern for markers of the G1-S phase transition of the cell cycle. The results of the present study support the concept of (a) an autoregulatory loop between pRB and cyclin D1 in tumours with a functional pRB and the disruption of this loop in the presence of pRB mutation, as well as (b) a checkpoint mechanism in late G1, whose regulation via loss of p16 or pRB, or overexpression of cyclin D1 constitutes a common pathway to malignancy. Further, the results raise the possibility of cyclin D1 overexpression having a role in the pathogenesis of uveal melanoma.

Figure 1

Interactions between cyclin D1 gene and protein (pD1) and retinoblastoma protein (pRB) as well as between the cyclin dependent kinases (CDKs) and their inhibitors (p16 and p21 via p53) during the cell cycle (modified from Lukas et al⁵⁵). During early to mid G1, the concentrations of pD1 are low. pRB is hypophosphorylated and has a stimulatory activity on the transcription of the cyclin D1 gene. At this time, the retinoblastoma "pockets" are occupied by "cellular retinoblastoma binding proteins" (CRBPs), which are potent cell cycle stimulators when released later from the pRB pockets. The CDKs are inhibited by the CDKIs, including p16 and p21, the latter being stimulated by p53. The combination of hypophosphorylated pRB, the pRB bound CRBPs, and the inhibitory effects of the CDKIs contribute to cell cycle arrest at the restriction point (R point) in the mid to late G1 phase. The concentrations of pD1 increase and eventually are sufficient to combine with the CDKs, overriding the inhibitory activity of the CDKIs and resulting in (a) the displacement of the CRBPs from the RB pockets, (b) phosphorylation of the pRB, causing a change in the configuration of the pRB molecule. These alterations stimulate cell progression into the S phase, as well as a decrease in the transcriptional stimulus of the cyclin D1 gene. The concentration of pD1, consequentially, decreases during the S, G2, and M phases. TF-X represents a proposed transcription factor⁵⁵ and BS, a possible binding site or molecule through which cyclin D1 indirectly interacts with pRB.

Figure 2

Normal retina with (a) cyclin D1 staining in occasional ganglion cells (arrow) (×20 objective); (b) p53 positivity in scattered ganglion cells (×40 objective); (c) granular p21 staining in all cell layers of the retina (×20 and ×40 objective); (d) BCL-2 positivity in glial cells and, at the higher magnification, in occasional ganglion cells (×20 and ×40 objective). (e) Normal choroid with scattered BCL-2 positive reactive lymphocytes (arrow) (×40 objective); occasional melanocytes demonstrated weak BCL-2 positivity following bleaching (not shown).

Figure 3

(a) Cyclin D1 positivity in reactive glial cells (arrow) within retinoblastoma; the tumour cells are negative (×20 objective). (b) The degree of differentiation within the retinoblastomas is reflected by the corresponding MIB-1 growth fractions (×20 objective); (c) p53; and (d) p21 positivity surrounding a pseudorosette where staining of these markers is present in all "zones" (×40 objective); (e) p16 staining in poorly and moderately differentiated tumours areas within a retinoblastoma (×20 objective). (f) Perivascular glial cells positive for BCL-2 in a retinoblastoma; the tumour cells are negative (×40 objective).

Figure 4

Cyclin D1 positivity in a spindle (a) and epithelioid (b) choroidal melanoma (×20 objective). pRB positivity in a spindle (c) and epithelioid (d) choroidal melanoma (×20 objective). (e) Clear p53 positivity in choroidal melanoma (arrow) (×20 objective). (f) p21 staining in occasional tumour cells of a mixed cell choroidal melanoma (×20 objective). (g) p16 positivity in scattered tumour cells of a mixed cell choroidal melanoma (×40 objective). (h) BCL-2 staining in a spindle choroidal melanoma (×20 objective).