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Addition of serum + inhibitors of protein synthesis

▲ FIGURE 21-29 General time course of expression of early- and delayed-response genes in G0-arrested mammalian cells after addition of serum. (a) In the absence of inhibitors of protein synthesis, expression of early-response genes peaks about 1 hour after addition of serum, which contains several mitogens, and then falls as expression of late-response genes begins. (b) Inhibitors of protein synthesis prevent the drop in expression of early-response genes and completely block expression of late-response genes. See text for discussion. [Adapted from A. Murray and T Hunt, 1993, The Cell Cycle: An Introduction, W. H. Freeman and Company.]

response genes. Mutant, unregulated forms of both c-Fos and c-Jun are expressed by oncogenic retroviruses (Chapter 23); the discovery that the viral forms of these proteins (v-Fos and v-Jun) can transform normal cells into cancer cells led to identification of the regulated cellular forms of these transcription factors.

After peaking at about 30 minutes following addition of growth factors, the concentrations of the early-response mRNAs fall to a lower level that is maintained as long as growth factors are present in the medium. Most of the immediate early mRNAs are unstable; consequently, their concentrations fall as their rate of synthesis decreases. This drop in transcription is blocked by inhibitors of protein synthesis (see Figure 21-29b), indicating that it depends on production of one or more of the early-response proteins.

Because expression of delayed-response genes depends on proteins encoded by early-response genes, delayed-response genes are not transcribed when mitogens are added to Go-arrested cells in the presence of an inhibitor of protein synthesis. Some delayed-response genes encode additional transcription factors (see below); others encode the D-type cyclins, cyclin E, CDK2, CDK4, and CDK6. The D-type cy-clins, CDK4, and CDK6, are expressed first, followed by cyclin E and CDK2 (see Figure 21-28). If growth factors are withdrawn before passage through the restriction point, expression of these G1 cyclins and CDKs ceases. Since these proteins and the mRNAs encoding them are unstable, their concentrations fall precipitously. As a consequence, the cells do not pass the restriction point and do not replicate.

In addition to transcriptional control of the gene encoding cyclin D, the concentration of this mid-G1 cyclin also is regulated by controlling translation of cyclin D mRNA. In this regard, cyclin D is similar to ,S. cerevisiae Cln3. Addition of growth factors to cultured mammalian cells triggers signal transduction via the PI-3 kinase pathway discussed in Chapter 14, leading to activation of the translation-initiation factor eIF4 (Chapter 4). As a result, translation of cyclin D mRNA and other mRNAs is stimulated. Agents that inhibit eIF4 activation, such as TGF-^, inhibit translation of cyclin D mRNA and thus inhibit cell proliferation.

Passage Through the Restriction Point Depends on Phosphorylation of the Tumor-Suppressor Rb Protein

Some members of a small family of related transcription factors, referred to collectively as E2F factors, are encoded by delayed-response genes. These transcription factors activate genes encoding many of the proteins involved in DNA and de-oxyribonucleotide synthesis. They also stimulate transcription of genes encoding the late-G1 cyclin (cyclin E), the S-phase cyclin (cyclin A), and the S-phase CDK (CDK2). Thus the E2Fs function in late G1 similarly to the S. cerevisiae transcription factors SBF and MBF. In addition, E2Fs autostimulate transcription of their own genes. E2Fs function as transcriptional repressors when bound to Rb protein, which in turn binds hi-stone deacetylase complexes. As discussed in Chapter 11, transcription of a gene is highest when the associated histones are highly acetylated; histone deacetylation causes chromatin to assume a more condensed, transcriptionally inactive form.

Rb protein was initially identified as the product of the prototype tumor-suppressor gene, RB. The products of tumor-suppressor genes function in various ways to inhibit progression through the cell cycle (Chapter 23). Loss-of-function mutations in RB are associated with the disease hereditary retinoblastoma. A child with this disease inherits one normal RB+ allele from one parent and one mutant RB~ allele from the other. If the RB+ allele in any of the trillions of cells that make up the human body becomes mutated to a RB~ allele, then no functional protein is expressed and the cell or one of its descendants is likely to become cancerous. For reasons that are not understood, this generally happens in a retinal cell leading to the retinal tumors that characterize this disease. Also, in most human cancer cells Rb function is inactivated, either by mutations in both alleles of RB, or by abnormal regulation of Rb phosphorylation. I

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