Carcinogen-transformed human cells are inhibited from entry into S phase by fusion to senescent cells but cells transformed by DNA tumor viruses overcome the inhibition.

Abstract

With parameters of transformation other than immortality (3). Another logical possibility is that the ability to induce DNA synthesis in senescent HDC might be associated with viral transformation. Cells transformed by viruses may have gained a factor that overrides the inhibitory effect of senescent HDC, whereas carcinogen-transformed cells may have lost a normal factor necessary for the expression ofsenescence while retaining their sensitivity to the putative inhibitor when it is supplied to them in heterodikaryons. We have tested these hypotheses by fusing senescent HDC to a series of transformed human cells that have various transformed properties and were transformed in different ways. MATERIALS AND METHODSOur procedures for cell culture, cell fusion, autoradiography, and identification of heterodikaryons have been described (2,3,5).In these experiments, the senescent HDC were either IMR-90 or WI-38 fetal lung fibroblasts at 48-53 population doublings. Forty-eight hours before fusion, senescent HDC were labeled with both [3H]thymidine (0.5 ,Ci/ml; 1 Ci = 3.7 X 10'°b ecquerels) and 2-,um-diameter latex beads (Dow The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation.

“…5), it is possible that the altered events in senescent HDF are clustered in just one or a few pathways that are deficient in senescent HDF. Prereplicative events that are altered in serum-stimulated senescent HDF include failure to show increased levels of c-fos (15) and cdc2, failure to phosphorylate RB protein (16), failure to abolish the inhibitor of entry into S phase as measured by the heterokaryon assay (22,36), and failure to enter S phase (Fig. 5).…”

“…(i) Unphosphorylated RB protein, a putative inhibitor of cell proliferation (39), is likely to be at least one component ofthe inhibitor of entry into S phase measured by the heterokaryon assay because only cells that contain RB-binding oncoproteins-i.e., SV40 T-antigen, adenovirus ElA, and HPV E7-are able to overcome the inhibitory effect of senescent HDF in heterokaryons (22 serum-stimulated, they go through a number of prereplicative steps before they enter S phase. All of the events illustrated are changes in gene expression except "PO4-RB," which means phosphorylation of RB protein, and "Abolish DNA Synthesis Inhibitor," which refers to loss of the ability to inhibit entry into S phase in replicating HDF in the heterokaryon assay (22,36). A majority of these prereplicative events still occurs in serum-stimulated senescent HDF even though these cells are unable to enter S phase (13)(14)(15).…”

“…However, in contrast to serum-stimulated quiescent HDF, serum-stimulated senescent HDF fail to express c-fas (15) and fail to phosphorylate the protein product of the retinoblastoma susceptibility gene (RB protein) (16) incubated for an additional 4 days in medium containing 0.1% serum. Senescent or quiescent cultures had <5% labeled nuclei following a 24-h period of [3H]thymidine incorporation (22).…”

Induction of senescence in human malignant glioma cells by p16INK4A

“…INK4A in inducing senescence in human head and neck keratinocytes revealed a correlation between the loss of p16 INK4A expression and keratinocyte immortality in several keratinocyte cell lines (Loughran et al, 1996) showed that the cell cycle block in senescent cells is not completely irreversible; when senescent ®broblasts were fused to SV40 transformed cells, DNA synthesis was induced in the senescent nuclei in the heterodikaryons (Stein et al, 1982), most likely as an eect of SV40 large T antigen. In senescent human diploid ®broblasts the p16…”

Expression of an EF-1α-like Rat cDNA, S1, in Escherichia coli and Production of a Rabbit Polyclonal Antiserum to the Recombinant Protein

“…(Stein et al, 1982). However, when senescent eclls were fuscd with immortal ccII lines containing DNA tumor virus, the immortal lines were able to overcome the inhibition of DNA synthesis.…”