Heterogeneous expression of melanoma antigen (hMAGE) mRNA in mesenchymal neoplasia.

Recommendations have been advanced recently for the use of cancer/testis (CT) immunotherapy against sarcomas. CT antigens are encoded by cancer-germline genes (e.g., hMAGE family) that are expressed in tumors and male germline cells but typically not in normal tissues. At present, little information is available regarding CT expression in mesenchymal neoplasms, and it remains uncertain whether CT immunotherapy will serve as a viable alternative or adjunct to current sarcoma therapies involving resection, followed by adjuvant radiotherapy and/or chemotherapy. In this study, hMAGEA2, hMAGEA3, hMAGEA4, and hMAGEC1 mRNA content in 21 benign mesenchymal tumors (representing seven histotypes) and 28 primary sarcomas (10 histotypes) was inventoried using real-time-PCR and then compared against hMAGE mRNA expression in non-sarcomatous malignancies, three cell lines, and muscle. hMAGEA2, hMAGEA3, and hMAGEC1 transcripts were infrequent in mesenchymal tissues in general, whereas hMAGEA4 mRNA was present in 84% of all mesenchymal tumors, 100% of non-sarcomatous tumors, all three cell lines, and in four of five muscle samples. Although hMAGEA4 mRNA was detected in four of five muscle preparations, there was no indication that the mRNA was translated into protein. The presence of hMAGEA4 mRNA in muscle, plus the inconsistent and infrequent occurrence of hMAGEA2, hMAGEA3, and hMAGEC1 mRNA within and among mesenchymal tumor histotypes, makes these four hMAGE antigens unlikely candidates for sarcoma-specific immunotherapy.

Expression of the superantigen Mycoplasma arthritidis mitogen in Escherichia coli and characterization of the recombinant protein.

Mycoplasma arthritidis mitogen (MAM), is a soluble protein with classical superantigenic properties and is produced by an organism that causes an acute and chronic proliferative arthritis. Unfortunately, the process of obtaining purified MAM from M. arthritidis culture supernatants is extremely time-consuming and costly, and very little material is recovered. Thus, our laboratory has expressed MAM in Escherichia coli by using a protein fusion expression system. The construction and expression of recombinant MAM (rMAM), as well as a comparison of the biological properties of rMAM to those of native MAM, are discussed. Briefly, conversion of the three UGA codons to UGG codons was required to obtain full-length expression and mitogenic activity of rMAM. Antisera to native MAM recognized both rMAM and the fusion protein. The T-cell receptor Vbeta and major histocompatibility complex class II receptor usages by rMAM and the fusion protein were identical to that of native MAM. In addition, the ability to induce suppression and form the superantigen bridge could also be demonstrated with rMAM. Importantly, dose-response experiments indicated that homogeneous native MAM and rMAM were of equal potency. Thus, MAM has been successfully expressed in E. coli, thereby creating a viable alternative to native MAM.

RIF-1 tumor treatment in anesthetized mice with minimal effects on blood flow and hypoxia.

The injectable anesthetic etomidate and a clip that facilitates hyperthermia by water bath immersion (the "Gibbs clip") were evaluated for their suitability with subcutaneous flank RIF-1 tumors in C3H/HeJ mice. For tumors between 100 and 250 mg (mean, 160 mg), etomidate at 40 mg kg-1 ip did not significantly increase the radiobiologic hypoxic fraction (RHF); as calculated from an in vitro assay after treatment in vivo the RHF increased from 0.06 (95% C.I.:0.03-0.13) to 0.08 (0.04-0.16). In contrast, for larger tumors (270-650 mg; mean, 400 mg) etomidate increased the RHF from 0.08 (0.04-0.17) to 0.28 (0.14-0.60). Holding 250-mg-or-less tumors 3-mm laterally away from the flank in an X-ray jig did not significantly reduce tumor blood flow as inferred from the clearance rates of Xe, but the RHF of 0.15 (0.08-0.26) was significantly (P less than 0.05) greater than the RHF in unanesthetized mice, although not the RHF in anesthetized mice. The Gibbs clip, which folded skin around a tumor to enhance thermal conduction from a water bath, did not impair the increase in tumor blood flow in response to the cardiovascular arousal associated with exposure to a hyperthermic stimulus. Intratumor temperature was within 0.25 degrees C of bath temperature 3 min after the tumor and clip were immersed, but only when rectal temperatures were at 37 degrees C or above; tumor blood flow increased intratumor temperature gradients by 0.10 degrees C for each 1.5 degrees C that the body temperature was below 37 degrees C.

In vitro response to hyperthermia or X-irradiation of diploid and tetraploid RIF-1 cells separated by centrifugal elutriation.

The RIF-1 cell line at the University of Utah is comprised of approximately 65 per cent diploid and 35 per cent tetraploid cells. Because sensitivity to heat cell killing has been shown to be ploidy dependent (Lucke-Huhle 1978), the responses of these subpopulations were examined independently. Diploid and tetraploid cells were separated from stock in vitro cultures by centrifugal elutriation and maintained entirely in vitro. No influence of ploidy on Do or Dq of heat dose survival curves could be detected. Neither did ploidy affect sensitivity to X-irradiation. However, separation of the diploid and tetraploid subpopulations was imperfect. The ratio of diploid to tetraploid cells in the separated and parent (mixed ploidy) lines was therefore monitored at each passage. Tetraploid contamination of the diploid cell subline was undetectable at the time of separation but regrew to 35 per cent by 40 days after separation. Diploid contamination of the tetraploid subline was initially less than 5 per cent and remained quite low until it became undetectable at 74 days. Differences in regrowth of the contaminating subline could not be accounted for by differences in plating efficiency or doubling time, but might result from subpopulation interactions. If so, such phenomena should be considered when using ploidy-dependent cytotoxic treatments.