The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Non-invasive detection of malignancy by identification of unusual CD44 gene activity in exfoliated cancer cells.

OBJECTIVE--To investigate non-invasive detection of cancer by testing for unusual CD44 gene activity in a clinical sample as an indicator of exfoliated tumour cells. DESIGN--Case-control study. SUBJECTS--44 unselected, consecutive patients with bladder cancer and 46 people with no evidence of neoplasia. MAIN OUTCOME MEASURE--Presence or absence of large CD44 gene products containing exon 6 derivatives in urine samples. RESULTS--Novel abnormalities in the pattern of expression of this gene, seen specifically in tumour tissue, led to cloning of a newly recognised coding region in it (exon 6). This was tested as a probe for detection of exfoliated malignant cells in naturally voided urine. CD44 gene products extracted from the urine and amplified with polymerase chain reaction contained predicted electrophoretic band of 735 base pairs in 40 of the 44 patients with bladder cancer (sensitivity 91%). Products from 38 of the 46 people with no evidence of neoplasia showed no such band (specificity 83%). CONCLUSIONS--Unusual activity of the CD44 locus in neoplasia and malignancy is confirmed, and techniques for the analysis of such activity can enable non-invasive investigation of patients for primary or recurrent bladder cancer or for other tumours that shed neoplastic cells into body fluids.     

The role of shaded cocoa plantations in the maintenance of epiphytic orchids and their interactions with phorophytes

生境丧失和破碎化是热带森林生物多样性的主要威胁。遮荫的可可种植园(SCP)等农业生态系统为热带森林生物群提供了庇护。然而,在这些转化后的生境中是否还维持种间生态的相互作用,目前尚鲜为人知。我们评定附生兰花群落的多样性、繁殖状态和光合代谢(CAM或C₃),以及与热带雨林(TRF)相比,它们与SCP中寄主树种(附生植物)之间的相互作用。在墨西哥东南部,对TRF和SCP中各三个采样地点进行研究。每个采样地点建立了4个400平方米的样地,调查记录所有兰花及其附生植物。我们依据花/果实(或残体)是否存在来确定每个兰花个体的繁殖(成体)或非繁殖(幼体)状态,并根据文献确定每种兰花的光合作用途径。我们采用真正的分集和生态网络的方法分别分析兰花的多样性以及兰科与附生植物间的相互作用。我们一共记录了47个兰花种的607个个体。在TRF (19个有效物种)中的兰花多样性高于SCP (11个有效物种),两个生境之间仅共享7个物种。SCP (53%)中的CAM兰花物种比TRF (14%)更常见。在群落水平上,SCP维持了非生殖兰花和生殖兰花的比例以及TRF兰科附生植物网络的嵌套结构和特异化水平。然而,SCP中仅保留一部分的TRF附生兰花,突显出保护TRF的重要性。尽管存在这种差异,诸如SCP类型的遮荫农业生态系统仍然可以维持天然林的一些多样性和功能,因为SCP附生兰花群落主要由CAM物种组成,其附生植物构成了一个嵌套的相互作用网络,对干扰形成了更强的抗性。     

Cancer-stroma targeting therapy by cytotoxic immunoconjugate bound to the collagen 4 network in the tumor tissue

Some cytotoxic immunoconjugates have been approved for malignant lymphoma, a representative of hypervascular and stroma-poor tumors. However, many human solid tumors possess abundant intercellular stromata that prevent diffusion of cancer cell-specific monoclonal antibodies (mAb) and become a barrier preventing immunoconjugates from directly attacking cancer cells. Here we show the successful development of a new strategy that overcomes this drawback and achieves a highly localized concentration of a topoisomerase I inhibitor, SN 38, by conjugating it via an ester bond to a mAb targeted against collagen 4, a plentiful component of the tumor stroma. Poly(ethylene glycol) (PEG) was utilized as a spacer, close to each bond, to maintain stability in the blood. Immunoconjugates selectively extravasated from leaky tumor vessels and minimally from normal vessels because the immunoconjugates are too large to pass through normal vessel walls. Stroma-targeting immunoconjugates bound to the stroma to create a scaffold, from which sustained release of cytotoxic agent occurred and the agent subsequently diffused throughout the tumor tissue to damage both tumor cells and vessels. Cancer-stroma-targeting immunoconjugate therapy was thus validated as a new modality of oncological therapy, especially for refractory, stromal-rich cancers.     

Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum

The aquatic leech, Theromyzon tessulatum, secretes a proteinaceous cocoon with extraordinary physical properties (e.g., proteolytic, thermal resiliency). The deduced amino acid sequence of a major protein (Tcp—Theromyzon cocoon protein) from the T. tessulatum cocoon wall has been used to model the endogenous structure of the Tcp protein. The Tcp protein sequence comprises six internal repeats, each containing 12 ordered Cys residues. Amino acid alignments suggest that the region Cys1→6 is homologous to antistasin, a leech anticoagulant, and Cys7→12 is homologous to an epidermal growth factor-like domain found in notch-class proteins, which play critical roles in development, signaling, and adhesion throughout the Animalia. Modeling of individual domains (i.e., antistasin and notch) positions multiple hydrophobic and charged residues on the surface. When the antistasin and notch domains were fused, hydrophobic pockets appeared that may facilitate a polymerization mechanism. Collectively, the predicted features of our Tcp model are consistent with the physical properties of the leech cocoon wall.      

Rabies Virus Envelope Glycoprotein Targets Lentiviral Vectors to the Axonal Retrograde Pathway in Motor Neurons

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.     

Metastasis-related Plasma Membrane Proteins of Human Breast Cancer Cells Identified by Comparative Quantitative Mass Spectrometry

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multistep process. The cancer cell proteins and plasma membrane proteins in particular involved in this process are poorly defined, and a study of the very early events of the metastatic process using clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice; but although one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane purification and comparative quantitative LC-MS/MS proteomics identified 13 membrane proteins that were expressed at higher levels and three that were underexpressed in the metastatic compared with the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5′-nucleotidase (CD73), NDRG1, integrin β1, CD44, CD74, and major histocompatibility complex class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, and immunocyto- and immunohistochemistry. Analysis of clinical breast cancer biopsies demonstrated a significant correlation between high ecto-5′-nucleotidase and integrin β1 expression and poor outcome, measured as tumor spread or distant recurrence within a 10-year follow-up. Further the tissue analysis suggested that NDRG1, HLA-DRα, HLA-DRβ, and CD74 were associated with the ER⁻/PR⁻ phenotype represented by the two cell lines. The study demonstrates a quantitative and comparative proteomics strategy to identify clinically relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy.Breast cancer is the most common malignant disease among women in Western countries, occurring in approximately one in 11 women (1). In this disease, malignant cells often disseminate to regional lymph nodes and establish distant metastases, preferentially in the bone, lung, and liver, resulting in poor outcome and high mortality (2, 3).Metastases are established through a complex set of events that is yet not fully understood but requires detachment of single cells from the primary tumor, penetration of the tissue matrix, and migration of these cells to distant locations where they induce angiogenesis and undergo expansive growth (4). Some disseminated cancer cells seem to be capable of maintaining dormancy in distant organs without establishing metastases but may suddenly become activated many years after resection of the primary tumor (5). The dormancy may be caused by environmental signals, either lack of those inducing differentiation or the presence of signals stimulating growth arrest. Cellular factors and changes in the microenvironment, such as inflammation or a change in hormonal status, might eventually induce proliferation, differentiation, and subsequent metastatic growth, whereas other disseminated cancer cells remain dormant for a lifetime (6).Traditional models of metastasis suggest that a subpopulation of cells in the primary tumor acquire metastatic capacity late in tumorigenesis, but gene expression profiles and cellular studies have recently provided evidence for a possible alternative model that suggests the metastatic capacity is acquired early in tumorigenesis (7). Stem cell populations have been identified in a range of hematopoietic and solid tumors and might represent the cells of origin for these tumors but might also be responsible for metastasis (8). Although a preserved genetic signature between the primary tumor and the metastasis has been found, other studies provide evidence of a gradual acquisition of genomic changes because distant metastases may not uniformly share mutations and often differ extensively from the primary tumor, reflecting the extent of genetic instability of breast cancer (9, 10). Only few studies provide proteomic characteristics of metastatic versus primary tumor of breast cancer because of the difficulties of obtaining high quality human tumor samples with full clinical histories and the absence of directly relevant in vitro assays (11, 12).The two isogenic cell lines M-4A4 and NM-2C5, which were derived from the MDA-MB-435 cell line and originated from a highly aggressive human invasive ductal carcinoma, provide an interesting model of the metastatic process (13). M-4A4 and NM-2C5, when inoculated into the mammary fat pad of nude mice, showed equal tumorigeneity, but although M-4A4 established easily detectable metastases restricted to lymph nodes and lungs, NM-2C5 cells disseminated to distal organs, but the cells remained dormant and did not establish metastasis (14). There is an ongoing debate on whether the parent cell line MDA-MB-435 can be defined as a breast cancer cell line because it, along with breast- and epithelia-specific markers, also expresses melanoma-specific genes (15). However, MDA-MB-435 can be induced to express breast differentiation-specific proteins and secrete milk lipids as observed in other well established breast cancer cell lines and has therefore been considered as an excellent model of a highly malignant and dedifferentiated breast cancer (16). Regardless of this debate, our model system remains valuable in the context of cancer metastasis, but the results should, as always when using cell line models, be supported by studies of clinically relevant human tissue specimens.M-4A4 and NM-2C5 have been extensively compared using gene expression analysis identifying a panel of differentially expressed genes (13, 17–20). However, because the proteome is so much more complex than the genome, similar studies at the protein level with special focus on plasma membrane proteins may add valuable biological insight and identify cell surface molecules that might be targeted with drugs or antibodies to inhibit the metastatic process.Comparative quantitative proteomics using stable isotope labeling with amino acids in cell culture (SILAC)¹ and LC-MS/MS allows a study of proteins with quantitatively different expression levels on metastasizing versus non-metastasizing cells. We used this technique to identify a panel of plasma membrane proteins showing altered expression in cells capable of forming metastasis. Validation studies at the protein and RNA expression level of the cell lines indicate that several of the identified proteins may be important for establishing metastasis in distant organs and thus have potential in target-specific therapy. Therefore, to further evaluate the clinical relevance of a selected number of the candidates identified by our analysis, their expression levels were evaluated in a panel of primary breast cancer biopsies and corresponding axillary lymph node metastasis from patients with known clinical outcomes. The results demonstrated the power of this systematic stepwise strategy for identifying targets of potential clinical value.     

Clinicopathological observations on metastasis in man studied in patients treated with peritoneovenous shunts.

Fourteen patients with inoperable cancer treated with peritoneovenous shunts for malignant ascites were studied post mortem. Clinical observations and findings at necropsy indicated that peritoneovenous shunting does not result in the establishment of clinically important haematogenous metastases and that metastases do not necessarily develop even when large numbers of viable tumour cells regularly enter the blood. Peritoneovenous shunting provides a unique opportunity for collecting data on the spread of tumours in man.