The hallmarks of cancer.

Mitogen-dependent progression through the first gap phase (G1) and initiation of DNA synthesis (S phase) during the mammalian cell division cycle are cooperatively regulated by several classes of cyclin-dependent kinases (CDKs) whose activities are in turn constrained by CDK inhibitors (CKIs). CKIs that govern these events have been assigned to one of two families based on their structures and CDK targets. The first class includes the INK4 proteins (inhibitors of CDK4), so named for their ability to specifically inhibit the catalytic subunits of CDK4 and CDK6. Four such proteins [p16 (Serrano et al. 1993), p15 (Hannon and Beach 1994), p18 (Guan et al. 1994; Hirai et al. 1995), and p19 (Chan et al. 1995; Hirai et al. 1995)] are composed of multiple ankyrin repeats and bind only to CDK4 and CDK6 but not to other CDKs or to D-type cyclins. The INK4 proteins can be contrasted with more broadly acting inhibitors of the Cip/Kip family whose actions affect the activities of cyclin D-, E-, and A-dependent kinases. The latter class includes p21 (Gu et al. 1993; Harper et al. 1993; El-Deiry et al. 1993; Xiong et al. 1993a; Dulic et al. 1994; Noda et al. 1994), p27 (Polyak et al. 1994a,b; Toyoshima and Hunter 1994), and p57 (Lee et al. 1995; Matsuoka et al. 1995), all of which contain characteristic motifs within their amino-terminal moieties that enable them to bind both to cyclin and CDK subunits (Chen et al. 1995, 1996; Nakanishi et al. 1995; Warbrick et al. 1995; Lin et al. 1996; Russo et al. 1996). Based largely on in vitro experiments and in vivo overexpression studies, CKIs of the Cip/Kip family were initially thought to interfere with the activities of cyclin D-, E-, and A-dependent kinases. More recent work has altered this view and revealed that although the Cip/Kip proteins are potent inhibitors of cyclin Eand A-dependent CDK2, they act as positive regulators of cyclin Ddependent kinases. This challenges previous assumptions about how the G1/S transition of the mammalian cell cycle is governed, helps explain some enigmatic features of cell cycle control that also involve the functions of the retinoblastoma protein (Rb) and the INK4 proteins, and changes our thinking about how either p16 loss or overexpression of cyclin D-dependent kinases contribute to cancer. Here we focus on the biochemical interactions that occur between CKIs and cyclin Dand E-dependent kinases in cultured mammalian cells, emphasizing the manner by which different positive and negative regulators of the cell division cycle cooperate to govern the G1-to-S transition. To gain a more comprehensive understanding of the biology of CDK inhibitors, readers are encouraged to refer to a rapidly emerging but already extensive literature (for review, see Elledge and Harper 1994; Sherr and Roberts 1995; Chellappan et al. 1998; Hengst and Reed 1998a; Kiyokawa and Koff 1998; Nakayama 1998; Ruas and Peters 1998).

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CDK inhibitors: positive and negative regulators of G1-phase progression

Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells have typically acquired damage to genes that directly regulate their cell cycles. Genetic alterations affecting p16(INK4a) and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein (RB) and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development. Like the tumor suppressor protein p53, components of this "RB pathway," although not essential for the cell cycle per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life.

http://science.sciencemag.org/content/sci/274/5293/1672.full.pdf

Cancer Cell Cycles

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Wnt/β-catenin signaling: components, mechanisms, and diseases

Deletions or mutations of the retinoblastoma gene, RB1, are common features of many tumors and tumor cell lines. Recently, the RB1 gene product, p105-RB, has been shown to form stable protein/protein complexes with the oncoproteins of two DNA tumor viruses, the adenovirus E1A proteins and the simian virus 40 (SV40) large T antigen. Neither of these viruses is thought to be associated with human cancer, but they can cause tumors in rodents. Binding between the RB anti-oncoprotein and the adenovirus or SV40 oncoprotein can be recapitulated in vitro with coimmunoprecipitation mixing assays. These assays have been used to demonstrate that the E7 oncoprotein of the human papilloma virus type-16 can form similar complexes with p105-RB. Human papilloma virus-16 is found associated with approximately 50 percent of cervical carcinomas. These results suggest that these three DNA viruses may utilize similar mechanisms in transformation and implicate RB binding as a possible step in human papilloma virus-associated carcinogenesis.

https://science.sciencemag.org/content/sci/243/4893/934.full.pdf

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product

Much has been written about the functions of the E2F transcription factor and the product of the retinoblastoma tumor suppressor gene (pRB). These proteins have been described in terms that vary from ‘‘master regulators of cell cycle and differentiation’’ to ‘‘peripheral factors that lie outside the core cell cycle machinery.’’ Most often, pRB and E2F are described in short and simple terms as opposing molecules that control the G1to Sphase transition. There is an element of truth in each of these descriptions. E2Fand pRB-family proteins clearly play important roles in cell proliferation and differentiation. The extent to which they are master regulators or peripheral factors is a question of semantics, and these terms tell us more about the writer than the proteins. Perhaps the most important development in the E2F literature is the appreciation that E2F and pRB are not unique molecules with functions that can be defined in black and white terms. Instead, E2F and pRB represent families of related proteins that have diverse and occasionally contradictory activities. We now know a great deal about E2F complexes and pRB-family proteins and the emerging picture defies a one-line explanation. The fascinating variety of activities ascribed to various E2F complexes challenges us to place these into context and to find the right perspective. This review is presented into two sections. The first section summarizes the tremendous progress into the composition and properties of E2F and the many interactions that coordinately regulate E2F-dependent transcription. The rapid growth in the size of the E2F literature hides the fact that several fundamental questions have not been fully answered. Because of this, the second section of this review details five unresolved issues that have been highlighted by recent publications. It is impossible to cover all of the relevant E2F literature in a single review and readers are referred to reviews by Farnham (1995); Sardet et al. (1997); Helin (1998); and Yamasaki (1998) for a comprehensive survey.

http://genesdev.cshlp.org/content/12/15/2245.full.pdf

The regulation of E2F by pRB-family proteins

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share significant amino acid sequence homology The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB) Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities The E7 protein of the bovine papillomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped Only a portion of the sequences that are conserved between the HPV E7 proteins and AdE1A were necessary for association with p105-RB Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1989.tb08594.x

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

Tumor Induction and Tissue Atrophy in Mice Lacking E2F-1

The E2 factor (E2F) family of transcription factors are downstream targets of the retinoblastoma protein. E2F factors have been known for several years to be important regulators of S-phase entry. Recent studies have improved our understanding of the molecular mechanisms of action used by this transcriptional network. In addition, they have given us an appreciation of the fact that E2F has functions that reach beyond G1/S control and impact cell proliferation in several different ways. The discovery of new family members with unusual properties, the unexpected phenotypes of mutant animals, a diverse collection of biological activities, a large number of new putative target genes and the new modes of transcriptional regulation have all contributed to an increasingly complex view of E2F function. In this review, we will discuss these recent developments and describe how they are beginning to shape a new and revised picture of the E2F transcriptional program.

Nicholas J. Dyson

Papers

The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product

The regulation of E2F by pRB-family proteins

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.

Tumor Induction and Tissue Atrophy in Mice Lacking E2F-1

The E2F transcriptional network: old acquaintances with new faces.