Interstitial collagenase MMP-1 and EMMPRIN in cell lines and in clinical specimens of cervical squamous cell carcinoma.

BACKGROUND: The aim of this study was to elucidate the features of the expression of matrix metalloproteinases inducer-EMMPRIN (EMN) and matrix metalloproteinase 1 (MMP-1) in cell lines and in clinical samples of cervical squamous cell carcinoma (SCC). MATERIAL AND METHODS: The study was carried out using RT-PCR, densitometry and immunohistochemical studies (IHC) on commercial cell lines Siha, Caski, transformed with HPV16; HeLa, and C33A transformed with HPV18, line C33A without HPV, and in clinical samples of SCC and morphologically normal tissue adjacent to the tumor. RESULTS: The data obtained indicate that the expression of mRNA EMN and MMP-1 occurs in all cell lines at different levels. HPV type and number of genes copies had no effect on expression degree both EMN and MMP-1. Gene expression of EMN and MMP-1 has been investigated in tumor and normal tissues. MMP-1 expression in tumor tissue in SCC, as a rule, has been significantly increased (2-6 times) compared to normal tissue. It was found in 90% of tumor samples. It is known, that MMP-1 promotes the development of invasive and metastatic processes. EMN expression was lower in the tumor tissue than in normal tissue in most cases. An increase in EMN expression was noted only in some cases of SCC. CONCLUSION: The data obtained indicate that MMP-1 can serve as a marker of the invasive potential of SCC. EMN, apparently, is not a major factor responsible for MMP-1 expression in SCC. Data are important for understanding the process of tumor development and may have prognostic value for the patient.

Role of mutations identified in ORFs M27, M36, m139, m141, and m143 in the temperature-sensitive phenotype of murine cytomegalovirus mutant tsm5.

A mutant of murine cytomegalovirus (MCMV), tsm5, which is temperature-sensitive for replication in murine embryo fibroblasts at 40°C, failed to replicate to detectable levels in mice. A total of 18 non-synonymous mutations have been identified in tsm5. In a previous study, a mutation (C890Y) identified in the M70 primase gene, when introduced into the wt M70 primase, resulted in a mutant with reduced viral replication at 40°C in vitro and which was severely attenuated in vivo. Five other previously identified mutations may also contribute to the tsm5 phenotype: (1) an A658S mutation in a protein expressed by the M27 ORF; (2) a V54I mutation in M36; (3) a Y565* mutation in m139; (4) a V195M mutation in m141; and (5) an M232I mutation in m143. In the present study, the above-mentioned mutations were introduced individually (M27, M36, m139, m141, m143) or together (M27/M36) into the MCMV K181 (Perth) variant bacterial artificial chromosome (BAC) using RecE/T homologous recombination. Growth in culture revealed that, apart from the double mutant (M27 and M36) and the m139 mutant, the introduced mutations in the above-mentioned genes did not show a temperature-sensitive phenotype in MEF or Raw 264.7 macrophage cells compared to their revertants or the wt virus. In contrast, replication of the M27/M36 double mutant was drastically reduced in MEFs at 40°C and in macrophages at 37°C. Replication of the m139 mutant was reduced in MEF cells at 40°C but not in macrophages. Thus, at least three further mutations contribute to the tsm5 phenotype.

Human Chr18 transcriptome dataset combined from the Illumina HiSeq, ONT MinION, and qPCR data.

The chromosome-centric dataset was created by applying several technologies of transcriptome profiling. The described dataset is available at NCBI repository (BioProject ID PRJNA635536). The dataset referred to the same type of tissue, cell lines, transcriptome sequencing technologies, and was accomplished in a period of 8 years (the first data were obtained in 2013 while the last ones - in 2020). The high-throughput sequencing technologies were employed along with the quantitative PCR (qPCR) approach, for data generation using the gene expression level assessment. qPCR was performed for a limited group of genes, encoded on human chromosome 18, for the Russian part of the Chromosome-Centric Human Proteome Project. The data of high-throughput sequencing are provided as Excel spreadsheets, where the data on FPKM and TMP values were evaluated for the whole transcriptome with both Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing.

Identification of mutations in a temperature-sensitive mutant (tsm5) of murine cytomegalovirus using complementary genome sequencing.

Identification of mutations in mutants derived chemically is a difficult and relatively random process. NimbleGen Comparative Genome Sequencing (CGS) was assessed as an inexpensive, rapid method of identifying mutations in the temperature-sensitive mutant tsm5 of the K181 (Birmingham) variant of murine cytomegalovirus (MCMV). This genome resequencing approach requires an established genome sequence as a reference. Comparison of tsm5 and the K181 (Birmingham) variant with the published K181 (Perth) MCMV genomic sequence revealed a total of 10 synonymous and 15 non-synonymous SNPs in tsm5 and 14 of the latter were confirmed by sequencing. Thus, while CGS cannot be relied upon to identify correctly all mutations it was helpful for identifying a large number of mutations for further investigation that could contribute to the ts phenotype of tsm5.

Role of mutations identified in ORFs M56 (terminase), M70 (primase) and M98 (endonuclease) in the temperature-sensitive phenotype of murine cytomegalovirus mutant tsm5.

Twenty-six non-synonymous and synonymous mutations have been identified in the temperature-sensitive (ts) mutant (tsm5) of the K181 (Birmingham) variant of murine cytomegalovirus that is deficient in DNA synthesis, processing and packaging at the non-permissive temperature and produces undetectable levels of infectious virus in mice. Non-synonymous mutations identified in the M70 (primase), M56 (terminase) and M98 (nuclease) ORFs were introduced individually and in combination into the K181 (Perth) variant using BAC technology to examine their role in the ts phenotype. The M56 (G439R) and M98 (P324S) mutations had no evident role in the ts phenotype. However, the C890Y M70 mutation alone and in combination with the M56 and/or M98 mutations rendered the virus ts, unable to replicate in mice and highly defective in DNA synthesis. Reversion of the tyrosine mutation to cysteine or introduction of C890M (experimentally) or C890S (naturally) restored the wt phenotype.

Ly49H+ NK cells migrate to and protect splenic white pulp stroma from murine cytomegalovirus infection.

In this study, we show that in the absence of a protective NK cell response, murine CMV causes destruction of splenic white and red pulp pulp areas in the first few days of infection. Destruction of T zone stroma is associated with almost complete loss of dendritic cells and T cells. We provide evidence that the virus replicates in red and white pulp stroma in vivo and in vitro. Control of white pulp viral replication is associated with migration of murine CMV-specific activated NK cells to white pulp areas, where they associate directly with podoplanin-expressing T zone stromal cells. Our data explain how NK cells protect the lymphoid-rich white pulp areas from CMV, allowing protective adaptive T cell-dependent immune responses to develop, and how this mechanism might break down in immunocompromised patients.