Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia

Refined cancer models are required if researchers are to assess the burgeoning number of potential targets for cancer therapeutics in a clinically relevant context that allows a fast turnaround. Here we use tumor-associated genetic pathways to transform primary human epithelial cells from the epidermis, oropharynx, esophagus and cervix into genetically defined tumors in a human three-dimensional (3D) tissue environment that incorporates cell-populated stroma and intact basement membrane. These engineered organotypic tissues recapitulated natural features of tumor progression, including epithelial invasion through basement membrane, a complex process that is necessary for biological malignancy in 90% of human cancers. Invasion was rapid and was potentiated by stromal cells. Oncogenic signals in 3D tissue, but not 2D culture, resembled gene expression profiles from spontaneous human cancers. We screened 3D organotypic neoplasia with well-characterized signaling pathway inhibitors to distill a clinically faithful cancer gene signature. Multitissue 3D human tissue cancer models may provide an efficient and relevant complement to current approaches to characterizing cancer progression.     

Systems analysis of primary Sjögren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model

Bone tissue has an exceptional quality to regenerate to native tissue in response to injury. However, the fracture repair process requires mechanical stability or a viable biological microenvironment or both to ensure successful healing to native tissue. An improved understanding of the molecular and cellular events that occur during bone repair and remodeling has led to the development of biologic agents that can augment the biological microenvironment and enhance bone repair. Orthobiologics, including stem cells, osteoinductive growth factors, osteoconductive matrices, and anabolic agents, are available clinically for accelerating fracture repair and treatment of compromised bone repair situations like delayed unions and nonunions. Preclinical and clinical studies using biologic agents like recombinant bone morphogenetic proteins have demonstrated an efficacy similar or better than that of autologous bone graft in acute fracture healing. A lack of standardized outcome measures for comparison of biologic agents in clinical fracture repair trials, frequent off-label use, and a limited understanding of the biological activity of these agents at the bone repair site have limited their efficacy in clinical applications.     

Targeting the cell cycle in breast cancer: towards the next phase

Deregulation of the cell cycle is a hallmark of cancer that enables limitless cell division. To support this malignant phenotype, cells acquire molecular alterations that abrogate or bypass control mechanisms in signaling pathways and cellular checkpoints that normally function to prevent genomic instability and uncontrolled cell proliferation. Consequently, therapeutic targeting of the cell cycle has long been viewed as a promising anti-cancer strategy. Until recently, attempts to target the cell cycle for cancer therapy using selective inhibitors have proven unsuccessful due to intolerable toxicities and a lack of target specificity. However, improvements in our understanding of malignant cell-specific vulnerabilities has revealed a therapeutic window for preferential targeting of the cell cycle in cancer cells, and has led to the development of agents now in the clinic. In this review, we discuss the latest generation of cell cycle targeting anti-cancer agents for breast cancer, including approved CDK4/6 inhibitors, and investigational TTK and PLK4 inhibitors that are currently in clinical trials. In recognition of the emerging population of ER+ breast cancers with acquired resistance to CDK4/6 inhibitors we suggest new therapeutic avenues to treat these patients. We also offer our perspective on the direction of future research to address the problem of drug resistance, and discuss the mechanistic insights required for the successful implementation of these strategies.     

Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation.

Proteolytic processing of the Rous sarcoma virus (RSV) Gag precursor was altered in vivo through the introduction of amino acid substitutions into either the polyprotein cleavage junctions or the PR coding sequence. Single amino acid substitutions (V(P2)S and P(P4)G), which are predicted from in vitro peptide substrate cleavage data to decrease the rate of release of PR from the Gag polyprotein, were placed in the NC portion of the NC-PR junction. These substitutions do not affect the efficiency of release of virus-like particles from COS cells even though recovered particles contain significant amounts of uncleaved Pr76gag in addition to mature viral proteins. Single amino acid substitutions (A(P3)F and S(P1)Y), which increase the rate of PR release from Gag, also do not affect budding of virus-like particles from cells. Substitution of the inefficiently cleaved MA-p2 junction sequence in Gag by eight amino acids from the rapidly cleaved NC-PR sequence resulted in a significant increase in cleavage at the new MA-p2 junction, but again without an effect on budding. However, decreased budding was observed when the A(P3)F or S(P1)Y substitution was included in the NC-PR junction sequence between the MA and p2 proteins. A budding defect was also caused by substitution into Gag of a PR subunit containing three amino acid substitutions (R105P, G106V, and S107N) in the substrate binding pocket that increase the catalytic activity of PR. The defect appears to be the result of premature proteolytic processing that could be rescued by inactivating PR through substitution of a serine for the catalytic aspartic acid residue. This budding defect was also rescued by single amino acid substitutions in the NC-PR cleavage site which decrease the rate of release of PR from Gag. A similar budding defect was caused by replacing the Gag PR with two PR subunits covalently linked by four glycine residues. In contrast to the defect caused by the triply substituted PR, the budding defect observed with the linked PR dimer could not be rescued by NC-PR cleavage site mutations, suggesting that PR dimerization is a limiting step in the maturation process. Overall, these results are consistent with a model in which viral protein maturation occurs after PR subunits are released from the Gag polyprotein.     

Focal adhesion complex proteins in epidermis and squamous cell carcinoma

Focal adhesions (FAs) are large, integrin-containing, multi-protein assemblies spanning the plasma membrane that link the cellular cytoskeleton to surrounding extracellular matrix. They play critical roles in adhesion and cell signaling and are major regulators of epithelial homeostasis, tissue response to injury, and tumorigenesis. Most integrin subunits and their associated FA proteins are expressed in skin, and murine genetic models have provided insight into the functional roles of FAs in normal and neoplastic epidermis. Here, we discuss the roles of these proteins in normal epidermal proliferation, adhesion, wound healing, and cancer. While many downstream signaling mechanisms remain unclear, the critically important roles of FAs are highlighted by the development of therapeutics targeting FAs for human cancer.     

香菇的学名及其在当代真菌分类中的位置

本文综述了香菇(Lentinula edodes)的分类历史,确认其在蘑菇目(Agaricales)Tricholomataceae科下的分类地位,并证实了它与多孔菌目(Poriales)Lentinaceae科的Lentinus属没有联系。根据《真菌、地衣汉语学名命名法规》,作者讨论了译为“香菇属”的Lentinus和“小香菇属”的Lentinellus两属的汉语学名问题,提出Lentinus的汉语学名应订正为“韧伞属”,Lentinellus为“螺壳菌属”。香菇所在的Lentinula属的汉语学名建议为“木菇属”。     

Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.     

Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens)

A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment- length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist.      

Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.