8 Mai 2017
Sperm protein 1. 7 is a novel cancer- testis antigen in multiple myeloma. Abstract. Various studies have demonstrated the aberrant expression of normal testicular proteins in neoplastic cells. These proteins collectively form the new class of tumor antigens called cancer- testis (CT) antigens. Their selective normal tissue expression makes them ideal antigens for immune targeting of the malignant disease.
In this study, the expression of a spermatozoa protein, Sp. It was found that Sp. Reverse transcription polymerase chain reaction (RT- PCR) and Western blot analysis detected Sp. Northern blot analysis and RT- PCR demonstrated that Sp. Since a high proportion of normal individuals develop antibodies against Sp. Sp. 17 is likely to be a highly immunogenic protein in vivo. Sp. 17 is therefore a novel member of the CT antigen family and should be an ideal target for immunotherapy of multiple myeloma.
Introduction. Therapeutic approaches for multiple myeloma remain a challenge. Although it is possible to induce disease remission in 5. Most patients die of disease relapse. Present therapeutic approaches aim to reduce the relapse rate by using maintenance chemotherapy or immunotherapy. Because immunotherapy is more specific and less toxic, it is an ideal approach, but at present there is a general lack of suitable candidate antigens.
The myeloma idiotypic protein is clone- specific and has been previously used. However, the clinical results have been disappointing, most likely owing to the weak immunogenicity of the idiotype proteins and low effector- to- target ratio generated by the vaccines.
Therefore, isolation and identification of other novel tumor antigens in myeloma will contribute to the ultimate development of a polyvalent vaccine that elicits consistent and strong immune responses and generates a high effector- to- target ratio in vivo. In this study, we demonstrated that sperm protein 1. Sp. 17) is a potential tumor antigen in myeloma. Both the transcripts encoding Sp.
Cancer testis antigens (CTA) are a large family of tumor-associated antigens expressed in human tumors of different histological. Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human J.
Study design. Materials. We studied 2 myeloma cell lines (ARP- 1 and ARK- B, gifts from J. Epstein, University of Arkansas for Medical Sciences, Little Rock, AK) and bone marrow from 1. The degree of bone marrow myeloma involvement ranged from 1. BB4 antibody–enriched myeloma cells were used). All clinical materials were obtained with patients' consent and with approval from the local ethics committee.
Rabbit polyclonal anti–human Sp. Michael O'Rand (The University of North Carolina at Chapel Hill, NC).
Reverse transcription- polymerase chain reaction. Total RNA was extracted from cells by means of the Tri- reagent (Sigma, St Louis, MO). All RNA specimens were first treated with DNAse I (Ambion, Austin, TX) to remove genomic DNA contamination. First strand c. DNA was synthesized from 1 . The polymerase chain reaction (PCR) primers were as follows: for 5. PCR was performed by means of 3.
Positive control amplification contained a plasmid with the Sp. DNA and a negative control of the PCR reaction mixture except for substitution of c. DNA by water. RNA integrity in each sample was checked by amplification of a GAPDH gene segment.
Successful removal of genomic DNA contamination was confirmed in each sample by amplification of the RNA without prior reverse- transcription (RT) reaction. PCR products were visualized on an ethidium bromide agarose gel for a DNA band of the expected size. All results were confirmed in 2 independent RT- PCRs. Northern blot analysis.
We electrophoresed 1. Subsequent hybridization to a 3. P- labeled probe (derived from a plasmid containing the full- length Sp.
DNA) and washing were performed under high- stringency conditions. Hybridization was performed at 6. Sp. 17 protein was detected by rabbit polyclonal anti- Sp. G antibody. Antibody binding was visualized by reaction with the Western blue stabilized substrate (Promega).
Flow cytometric analysis. This was carried out on myeloma cell lines by means of a FACScan. Rabbit polyclonal anti- Sp. Results and discussion. In addition to idiotypes, T- lymphocyte targets on myeloma cells that may be suitable molecules for immunotherapy include MUC- 1,7 mutant ras oncogene protein,8 and the new class of tumor antigens, known as CT (cancer- testis) antigens. The CT antigens include members of the MAGE family, BAGE, GAGE, and.
NY- ESO- 1. They are normal testicular antigens expressed aberrantly in tumor cells. Their restricted normal tissue expression makes them ideal molecules for immune targeting. CT antigens are expressed in some myeloma cells. Anti–MAGE- A3 cytotoxic lymphocyte clones raised from normal healthy donors could lyse myeloma cells in an HLA- A1– and HLA- A2–specific manner.
There are a number of criteria that a protein should fulfill to be an ideal tumor antigen for immunotherapy. In addition to being expressed by tumor cells, the antigen must be immunogenic in vivo and show limited normal tissue expression that would make the T- cell targeting safe. To address these points, we have investigated in this study the expression of Sp. CT antigen that may be suitable for immunotherapy of myeloma. Sp. 17 is a protein of apparent molecular weight of 2. It has, in the last few years, been the target of investigation as an immunocontraceptive.
Sp. 17 was chosen for this study because of its potential tissue specificity. In addition, it is likely to be a highly immunogenic protein in vivo on the basis of previous works showing a high incidence of auto- antibodies against Sp.
Both B- and T- cell responses against Sp. Using a pair of sequence- specific primers, we first demonstrated the presence of Sp.
RNA (m. RNA) in 2 out of 2 myeloma cell lines and 7 of 4. RNA samples from fresh unfractionated myeloma bone marrow (Figure 1. A). In another 6 bone marrow specimens obtained from myeloma patients, myeloma cells were purified with the use of the BB4 antibodies (directed at syndecan- 1 expressed on myeloma cells); Sp. In contrast, Sp. 17 m. RNA was not detected in the bone marrow from any of the 1. Fisher exact test, P = .
To confirm that the Sp. RNA resulted in the production of the Sp. BB4 antibody–purified fresh myeloma cells and ARK myeloma cell lines for Western blot analysis. Sp. 17 protein was detected in all of the 5 BB4- enriched specimens that were also positive by RT- PCR and in the ARK cell line but not detected in either of the 2 normal bone marrows (Figure 1. B). Our results therefore suggest that Sp. RNA and the protein level, in myeloma cells but not in normal bone marrow.
It is likely that the prevalence of Sp. RT- PCR of total RNA derived from unfractionated bone marrow because of the relative low m. RNA copy number within the myeloma cells that are actively synthesizing high levels of idiotypic protein. The detection may, however, be increased by enrichment of the tumor cell population. Analysis showing that Sp. A) PCR analysis using a pair of sequence- specific primers for Sp.
Lane 1 shows Sp. 17- positive myeloma bone marrow; lane 2, Sp. M, molecular marker. Lane i shows PCR of DNAse I- treated RNA; lane ii, PCR of RNA that had undergone RT; lane iii, control amplification for GAPDH gene segment. Lanes 1 through 6 show lysates from fresh myeloma cells; lane 7, lysate from ARK- B myeloma cell line; lanes 8 and 9, lysates from normal bone marrow. Panel i shows a strong signal, of approximately 1. Northern blot analysis; panel ii, 2. S ribosomal RNA; panel iii, RT- PCR showing Sp.
RNA; panel iv, control amplification for GAPDH gene segment. Since tissue specificity is a vital consideration in the choice of an antigen for immunotherapy, we proceeded to determine the expression of Sp. RNA. Positive signals of approximately 1. Figure 1. C). By RT- PCR, we also excluded low copy number expression in these normal tissues (Figure 1. C) and showed that the transcripts were detected only in normal testis, confirming the restricted normal tissue distribution of the Sp. Although the PCR was not designed primarily to give accurate m.
RNA quantitation, the reproducibly weaker signal from Sp. PCR products suggests a lower level of Sp. Figure 1. C). Sp. T- cell targeting in myeloma. Sp. 17 is also expressed on the 2 myeloma cell lines as a surface protein (Figure. Expression of Sp.
The expression of Sp. ARP- 1, upper panel; ARK- B, lower panel) is demonstrated by flow cytometry (2- stage cell- staining technique).
Rabbit Sp. 17 polyclonal antiserum was used to stain the cells. Phosphate- buffered saline was used as a control. In conclusion, our findings support the suitability of Sp.
Sp. 17 is a new member of the class of CT antigens. Work is ongoing to use Sp.
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