The PrPs in the tissues of the malignant tumors show different electrophoretic patterns in Western blots compared with that of brain tissues
To see the presence and the electrophoretic profiles of the PrP proteins in the malignant tissues, three randomly selected prepared tissue homogenates of each type of cancers were pooled and subjected into Western blots with PrP mAb 3F4. In parallel, the brain homogenates from a sCJD case and a normal donator died of car accidence, as well as that of scrapie agent 263K-infected hamster were loaded as the control. Clear three PrP specific bands were observed in the preparations of brain specimens, which were at the positions from 25 to 35 kDa representing di-, mono- and non-glycosylated PrPs (Fig 1). Additionally, larger molecular weights of PrP-specific signals were also detected in the brain homogenates of sCJD and 263-infected hamster. In contrast, the pooled samples of cancer tissues displayed the distinguishing electrophoretic patterns, containing more positive blotted bands (Fig 1). The most predominate bands were larger molecular weight signals, which migrated at almost the same position as those in the brain homogenates. There was relatively weak but clear positive band at the position of monoglycosylated PrP in all tumor preparations, and the bands at the position of aglycosylated PrP in some preparations, but no clear band corresponding to diglycosylated PrP (Fig 1). Additionally, the PrP reactive patterns in Western blot of all tested cancers seemed to be similar and the amounts of PrP positive signals were also undistinguished after digital assays of the gray values of all PrP signals (Fig 1).
To further assess the glycosylating features of PrP signals in tumor tissues, the homogenates of three laryngeal carcinomas and two benign polys were undergone into the process of deglycosylation together with or without PK digestion. In the reaction of PNGase F without PK treatment (Fig 2A, middle panel), all tested laryngeal samples revealed distinct PrP signals at the position of 25 kDa approximately, which migrated at the same position as the brain samples of healthy or diseased human and hamster. Besides, a weak and small molecular signal was also observed in some malignant tissues, which looked to be slightly higher in SDS-PAGE than those of brain tissues. In the preparation of PNGase F after 20 μg/ml PK-digestion (Fig 2A, bottom panel), as expected, the PrP signals in the normal human and hamster brain samples disappeared and those in sCJD case and 263K-infected hamster shifted to the position slightly higher of 20 kDa. PrP specific signals were still detectable in the three tested malignant cancers that seemed to be still slightly higher than those in TSE brain samples. No or very faint PK-resistant PrP signal was observed in the homogenates of two benign laryngeal polys.
Subsequently, the immunoreactivities of PrP signals in tumor tissues were tested by Western blots with various PrP specific antibodies. The recognizing peptides within PrP of those antibodies were illustrated in Fig 2B (upper panel). The pooled samples from three gastric cancers and the pooled samples of the pericarcinous tissues from the same three cases were subjected into the tests, using the brains of 263K-infected hamster as the control. As shown in Fig 2B, two predominate PrP bands were observed in all reactions, one was large molecular weight signal and the other was at the position of monoglycosylated PrP. In addition to the difference in signal strength, the reactive profiles to various PrP antibodies looked to be similar. Meanwhile, PrP reactive profiles in carcinous and pericarcinous tissues were also quite comparable. It indicates that the PrPs in the cancer tissues are probably full-length ones.
There is no significant difference in PrP amount evaluated by Western blots between carcinous and pericarcinous tissues
The changes of PrP in various kinds of human malignant tissues are widely described, among them most are based on immunohistochemical assays. To evaluate the potential difference in PrP amount in Western blots between the carcinous and pericarcinous tissues, different numbers of gastric, colon, liver, lung, thyroid and laryngeal cancers were employed into Western blots with mAb 3F4, together with the individual pericarcinous tissues in parallel. In general, the PrP reactive patterns were similar between carcinous and pericarcinous tissues (Fig 3). The signal intensity among the different cases in some special types of cancers varied. Quantitative assays of the average gray values of total PrP signals after normalized with the individual actin showed slightly higher in the carcinous tissues of gastric (Fig 3A), liver (C), thyroid (E) and laryngeal (F) cancers, while slightly higher in the pericarcinous tissues of colon (B) and lung (D), but without statistical significance. It highlights that under our experimental condition, either PrP reactive profiles or intensities in the carcinous and pericarcinous tissues are undistinguishable by the Western blots.
The PrPs in the carcinous and pericarcinous tissues are PK-sensitive, but vary among the tissue types
To test the features of PK-resistance of PrPs in various tumors, the pooled carcinous and pericarcinous samples consisting of randomly selected three cases were prepared. After exposed to different concentrated PK for 1 hr, the digestions were stopped and subjected into Western blots immediately. As shown in Fig 4, the PrP signals in all tested tissue kinds of carcinous and pericarcinous samples were reduced in dose-dependent manner. PrPs in all types of the tissues were partially but clearly PK resistant to the digestion of 20 μg/ml. At the same PK working concentration (20 μg/ml), the PrP signal in the normal brain homogenates vanished completely (data not shown). Along with the increase of PK amount, the PrP signals of large molecular weight completely disappeared, whereas those of small molecular weight were remarkably weaker and eventually undetectable. Different tissue types seemed to show slight diversity of PK-resistances. Quantitative measures of the average gray values revealed that PrP signals in gastric (Fig 4 A) and colon (D) tissues reduced to 20-60% in the reaction of 20 μg/ml, and less than 7% in the reaction of 50 μg/ml compared with that without PK. The PrP signals in the tissues of liver (C), lung (D) and thyroid (E) seemed to be more sensitive to PK digestion, which reduced to almost undetectable in the preparation of 50 μg/ml PK. The PrP signals in laryngeal tissues (F) were markedly more PK-resistant, which maintained 70% signals in the reaction of 20 μg/ml and about 25% in that of 50 μg/ml PK. Comparison of the PK-resistance of PrP signals in carcinous and pericarcinous tissues proposed a tendency that the PrP signals in malignant tissues had slightly stronger tolerance to PK digestion. Those data indicate that the PrPs in the malignant tumors are generally PK sensitive, despite of slightly more tolerant to PK digestion than the PrPs in brains.