Parkin Somatic Mutations Link Melanoma and Parkinson's Disease

Epidemiological studies suggest a direct link between melanoma and Parkinson's disease(PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28 K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.     

Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer-derived mammary tumor cells in suspension

Summary The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY-beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incoerporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in susfpension resulted in 10- to 400-fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Incrases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 μM hydrocorticone further increased MMTV yield two-to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure. This work was supported by the T. J. Martell Foundation for Cancer and Leukemia Research and by USPHS grant 5P-30CA23102. F. M. is a trainee on MSTP grant GM07280 from the National Institute of Health. This work was submitted in partial fullfillment of the requirements for the Ph. D. degree (F. M.).     

Cloned mouse mammary tumor virus DNA is biologically active in transfected mouse cells and its expression is stimulated by glucocorticoid hormones

We have cloned circular unintegrated mouse mammary tumor virus (MMTV) DNA from infected rat hepatoma cells in bacteriophage lambda. Seven independent clones containing MMTV DNA of homogeneous length of 9 kb (five) or 10 kb (two) were identified. The five 9 kb clones had identical restriction maps consistent with that of 9 kb unintegrated DNA; the other two were aberrant. MMTV DNA inserts were purified, ligated and used for cotransfection of Ltk^? cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. All Tk⁺ cell clones acquired new MMTV sequences and those transfected with the 9 kb MMTV DNA synthesized normal viral RNA and proteins. Viral gene expression was increased by the addition of dexamethasone.     

Identification of three human sequences with viral superantigen-specific primers

The open reading frame (ORF) in the long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) has recently been shown to encode multiple products including a negative acting factor (Naf) and a superantigen (Sag). Expression of superantigens from endogenous MMTV loci in the mouse results in the deletion of whole classes of T cells. In a PCR approach, with primers to the MMTV ORF and hybridization to MMTV specific probes, we have identified three human sequences. Direct sequencing of PCR products revealed that one of these products is related to a human autoantigen that is conserved among many species and is expressed in testes and sperm. The second sequence that we have identified is novel, and no evidence for expression of this sequence could be obtained. Finally, the third ORF-like sequence is a new member of a previously described family of human endogenous retroviruses (RTVL-I). This sequence is transcribed in several human cell lines, including B lymphoblastoid cells, and is thus the first demonstration that an RTVL-I-related sequence can be expressed. Taken together, these findings raise the intriguing possibility that the human genome contains superantigen-like sequences, some of which are also related to endogenous retroviruses, that may influence the T cell repertoire.     

Identification of the segments of the mouse transferrin receptor 1 required for mouse mammary tumor virus infection

Most enveloped viruses enter cells through binding of virion surface envelope proteins to receptors found on the plasma membrane of the cell. The beta retrovirus mouse mammary tumor virus (MMTV) uses transferrin receptor 1 (TfR1) to enter cells in a pH-dependent mechanism, probably co-trafficking with TfR1 to an acidic compartment where virus entry occurs. We have shown here that, although mouse and rat TfR1 function as entry receptors, cat, dog, hamster, or human TfR1s do not support MMTV infection. We also demonstrated that MMTV entry is independent of transferrin, iron, and the TfR1 cofactor hereditary hematochromatosis HFE protein. Using chimeric mouse/human hybrid TfR1 constructs, we determined the site of interaction with MMTV and found that it maps to two segments physically disparate from the TfR and HFE binding sites. Thus, MMTV has apparently evolved to enter cells independently of the iron status of the host.     

Identification of the receptor binding domain of the mouse mammary tumor virus envelope protein

Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe(40) to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe(40) residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation.     

Genetic identification of the pore domain of the OmpC porin of Escherichia coli K-12.

We have isolated and characterized 31 mutations in the ompC gene which allow Escherichia coli to grow on maltotriose (Dex+) in the absence of the LamB and OmpF porins. These ompC(Dex) mutations include single-base-pair substitutions, small deletions, and small insertions. DNA sequence analysis shows that all of the alterations occur within the coding region for the first 110 amino acids of mature OmpC. The 26 independent point mutations repeatedly and exclusively alter residues R37, R74, and D105 of mature OmpC. In each case, a charged amino acid is changed to an uncharged residue. Biochemical and physiological tests suggest that these alterations increase the size of the pore channel. Starting with three different ompC(Dex) strains with alterations affecting R74, we isolated mutants that could grow on maltohexose (Hex+). These mutants each contained a second alteration in the ompC gene involving residues R37, D105, or R124. The combined effects on pore function of the two mutations appear to be additive. These experiments suggest that we have identified the important residues of OmpC peptide involved in pore function. On the basis of these mutations and general rules for membrane protein folding, a model for the topology of the OmpC protein is proposed.     

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.     

Isolation of a pathogenic clone of mouse mammary tumor virus.

Exogenous mouse mammary tumor virus (MMTV) was cloned from a GR mammary tumor. Clone lambda GRT39 contained a full-length integrated MMTV(GR) provirus and both 5' and 3' host flanking DNA. The lambda GRT39 provirus had no apparent structural changes associated with cloning and retained the exogenous MMTV gag gene poison sequence. When introduced into rat mammary adenocarcinoma LA7 cells, the lambda GRT39 provirus was fully expressed. lambda GRT39-transfected LA7 cells made MMTV RNA, had gp52 SU protein on the cell surface, and produced B-type retrovirus particles characteristic of MMTV. Mammary tumors developed in hormone-stimulated BALB/c females injected with MMTV from lambda GRT39-transfected LA7 cells [MMTV (lambda GRT39)]. The tumors had new, clonally integrated copies of the MMTV(lambda GRT39) provirus and were expressing MMTV antigen. These data indicate that the lambda GRT39 provirus is biologically active and pathogenic.     

Hormonal regulation of mouse mammary tumor growth

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.     

抗体选择压作用下新城疫病毒HN和F基因的演化及其抗原性变异的比较分析

TZ060107株新城疫病毒(NDV)在含有对它抗体的鸡胚成纤维细胞(CEF)培养上分3个独立系列连传50代,每10代扩增其HN和F基因并测序。选择变异最大的系列A1-50病毒,再在含有抗A1-50抗体的CEF培养上分3个独立系列连续传50代,同时设3个不带抗体的独立传代系列作为对照。对第60、70、80、90、100代病毒的HN和F基因序列比较结果显示,有抗体组HN基因的非同义突变(NS)对同义突变(S)比值NS/S为5.25,明显高于无抗体组NS/S的2.375。前50代在抗体选择压作用下已发生的稳定NS突变在含有抗A1-50抗体的细胞培养中传代仍能稳定保持,且又出现了一个新的稳定的NS突变位点。在有抗体组经传50代后F基因发生的稳定非同义突变,在抗A1-50血清作用下再连传50代后也仍然保持,且又出现3个新的稳定的NS突变。不同传代病毒与原始病毒间的血清交叉血球凝聚抑制试验结果表明,随着在含有抗NDV血清的细胞培养上传代代数的增加,病毒与原始病毒间在抗原性的差异越来越大。