Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFß1 from Breast Cancer Patients.

BACKGROUND: Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor ß1 (TGFß1) in BC patients. RESULTS: To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFß1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN/nALN results revealed overexpression of both nuclear cFOS and cytosolic TGFß1 protein levels. However, only the TGFß1 data exhibited statistically significant overexpression, which was proportional to the respective values of mALN diameter of tumor deposits. CONCLUSIONS: Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

Additional confirmation of successful cell suspension fractionation of metastatic axillary node tissue.

We present herein an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients.

Identification of differentially expressed mRNA transcripts in drug-resistant versus parental human melanoma cell lines.

BACKGROUND: Malignant melanoma resistance to chemotherapy remains a major limitation to treatment. Our aim was to identify genes associated with drug resistance, in order to better understand the molecular events underlying the drug-resistant phenotype. MATERIALS AND METHODS: A human melanoma cell line and its drug-resistant variants obtained by selection with MNNG or 6-thioguanine were used. Alterations in gene expression were characterized by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Prominent mRNA fragments present in selected variants and not in the parental cells were identified and characterized by cloning and sequencing. Differential expression was confirmed by real-time RT-PCR. RESULTS: Three functionally distinct transcriptional products were demonstrated: the chaperonin subunit TCP 1-zeta-6A (CCT6A), the hyaluronan receptor CD44 and LPPR-2, the lipid phosphate phosphatase-related protein type-2. CONCLUSION: Genes with altered expression were identified in drug-resistant variants. The identified molecules may provide new insights into the molecular basis for melanoma resistance to chemotherapy.

Case with triple-negative breast cancer shows overexpression of both cFOS and TGF-ß1 in node-positive tissue.

We present herein a case report style article on a rare advanced triple-negative breast cancer (TNBC) patient with 6-month disease-free interval, and 10-month overall survival. Our results demonstrate that the poor clinical outcome of this patient was associated with pronounced, more than fivefold higher, overexpression of both cFOS and TGF-ß1 proteins in its metastatic nodal tissue extracts, when compared with the values of the two non-TNBC controls (with 'zero' disease-free interval and overall survival). This original observation suggests, for the first time, that both the cFOS and TGF-ß1 may be considered as a pair of biomarkers for an early assessment of poor prognosis for TNBC patients. The possible clinical implication of this observation is discussed.

Genomic instability in drug-resistant human melanoma cell lines detected by Alu-I-arbitrary-primed PCR.

Destabilization of the genome seems to be an important step in the generation of drug resistance. Since malignant melanoma is extremely resistant to chemotherapy, we used human melanoma cell lines as a model to investigate the putative role of genomic instability in the appearance of drug resistance. Drug-resistant variants were obtained with MNNG, BiCNU, doxorubicin and 6-thioguanine selection of melanoma cell lines. Genomic alterations in variant cells were detected by arbitrarily primed PCR of Alu-I digested DNA (Alu-I-AP-PCR). Two differential DNA bands from 6-TG-resistant cell variants were sequenced. One is homologous to intron 25 of the neural cell adhesion molecule L1 and the second to endogenous retroviral LTR sequences. We have shown that drug-resistant melanoma cell lines accumulate genomic alterations that are efficiently detected by Alu I-AP-PCR and that drug-resistant variants show genomic instability, including variations in LTR sequences, which may be associated with the appearance of the drug resistance phenotype.

Quantification of transforming growth factor beta 1 levels in metastatic axillary lymph node tissue extracts from breast cancer patients: a new specimen source.

OBJECTIVE: To use cytoplasmic tissue extract as a new specimen source to quantify transforming growth factor beta 1 (TGFbeta1) protein in metastatic axillary lymph node tissue (ALNT) of breast cancer (BC) patients and to confirm the feasibility of this approach in a prospective pilot study on a subgroup of patients with invasive BC. STUDY DESIGN: The 6 selected malignant and autologous nonmalignant pairs of ALNT were fractionated, under special preanalytical, nonaggressive/nondenaturing conditions, to obtain respective cytoplasmic extracts for TGFbeta1 detection by the Quantikine (R&D Systems Inc., Minneapolis, Minnesota, U.S.A.) enzyme-linked immunosorbent assay kit. RESULTS: The data indicated a highly significant (r = 0.973054) positive linear correlation between the TGFbeta1 concentration and total protein concentration in cytoplasmic extract of metastatic ALNT. The subsequent patients' pilot study, performed strictly before any clinicopathologic factors were accessible, revealed significantly (p < 0.01) elevated TGFbeta1 in malignant ALNT (median value: 1.05 ng/mg protein, range: 0.67-3.6 ng/mg protein, n = 6) vs. autologous nonmalignant ALNT controls (median value: 0.48 ng/mg protein, range: 0.29-0.90 ng/mg protein, n = 6). This elevation was correlated with the number of metastatic axillary lymph nodes with respect to the total and was consistent with an increase in size of tumor deposits in axillary lymph nodes. CONCLUSION: Our data provide for the first time suggestive evidence that the TGFbeta1 level in cytoplasmic extracts of metastatic ALNTs may be a promising biomarker of invasiveness for BC patients. Confirmatory, large-scale studies are needed to evaluate possible implications of this putative biomarker in BC diagnosis and treatment.

Differential gene expression in yellow-necked mice Apodemus flavicollis (Rodentia, Mammalia) with and without B chromosomes.

Most B chromosomes are heavily heterochromatic, promoting the general idea that they are genetically inert. The B chromosomes of Apodemus flavicollis are euchromatic and show a high degree of homology with the A chromosomes. The euchromatic nature of B chromosomes in A. flavicollis suggests that they may carry active genes and have transcriptional activity. We applied the differential display reverse transcription-polymerase chain reaction (DD RT-PCR) in order to analyze and compare gene expression in animals possessing B chromosomes and animals without B chromosomes. After a second and third round of amplification, three cDNA fragments were differentially expressed in +B mice compared with 0B animals. These cDNAs were Chaperonin containing TCP-1, subunit 6b (zeta) (CCT6B), Fragile histidine triad gene (FHIT) and hypothetical gene XP transcript. The differential expression pattern was confirmed by Real Time RT-PCR. We suggest that altered expression of these important genes is due to the presence of B chromosomes. In elevating the expression of these genes, B chromosomes of A. flavicollis affect some of the crucial processes in the cell. The significance of these effects and the nature of B chromosomes of A. flavicollis are discussed in the context of the data presented.