MUC1 isoform specific monoclonal antibody 6E6/2 detects preferential expression of the novel MUC1/Y protein in breast and ovarian cancer.

The products of the MUC1 gene are known to be highly expressed in human breast cancer cells. The best characterized MUC1 protein is a polymorphic, type 1 transmembrane molecule containing a large extracellular domain composed primarily of a variable number of 20 amino acid tandem repeats. We have recently identified a novel protein product of the MUC1 gene, the MUC1/Y protein, that is also a transmembrane protein but is devoid of the tandem repeat array and its immediate flanking sequences. To analyze its expression in tumor cells we generated monoclonal antibodies directed against the MUC1/Y extracellular domain (anti-MUC1/Yex MAbs). Epitope mapping identified the MAb, 6E6, which recognized the MUC1/Y isoform with exquisite specificity- the repeat-array-containing MUC1 isoform could not compete out this immunoreactivity. A 30mer peptide which is unique for MUC1/Y and corresponds to the "join" region generated by the MUC1/Y specific splice, abrogated all 6E6 MAb immunoreactivity towards MUC1/Y. Immunoprecipitation of the MUC1/Y protein with 6E6 MAbs revealed that, in contrast with the proteolytic cleavage of the tandem-repeat-array-containing MUC1 isoform, MUC1/Y is not cleaved. Flow cytometry analyses using the 6E6 MAbs demonstrated that the MUC1/Y isoform is expressed on the cell surface of both MCF-7 breast cancer cells and malignant epithelial cells present in effusions obtained from breast and ovarian cancer patients. Our results unequivocally establish that the MUC1/Y protein is expressed on the surface of breast cancer cells and cells of other epithelial malignancies. The anti-MUC1/Y MAbs described here can target MUC1/Y expressing tumor cells in vivo and are likely to be important reagents both for epithelial tumor diagnosis and immunotherapy.

The breast cancer-associated MUC1 gene generates both a receptor and its cognate binding protein.

MUC1 proteins, some of which contain a mucin-like domain and others lacking this region, can be generated from the human breast cancer-associated MUC1 gene by alternative splicing. The MUC1/Y isoform is devoid of the mucin domain and is a cell membrane protein that undergoes transphosphorylation on both serine and tyrosine residues. We have identified cognate binding proteins that specifically interact with the extracellular domain of MUC1/Y. Coimmunoprecipitation analyses clearly revealed the presence of complexes composed of MUC1/Y and its cognate binding proteins in primary breast tumor tissue. MUC1/Y-expressing mammary tumor cells can be specifically targeted, in vivo, with the labeled cognate binding protein. The k(D) of MUC1/Y for its binding proteins was estimated as 1.2 nM. The MUC1/Y binding proteins are also derived from the MUC1 gene and represent the secreted mucin-like polymorphic MUC1 proteins MUC1/SEC and MUC1/REP, which contain a tandem repeat array. Whereas nonposttranslationally modified MUC1/Y bound efficiently to MUC1/SEC, the latter mucin-like protein had to be posttranslationally modified in a cell-type specific manner to bind MUC1/Y. The interaction of MUC1/Y with MUC1/SEC has important biological functional correlates: (a) it induces MUC1/Y phosphorylation; and (b) it has a pronounced effect on cell morphology. These findings suggest that MUC1/Y and MUC1/SEC form an active receptor/ cognate binding protein complex that can elicit cellular responses. The proteins comprising this complex are, thus, generated by alternative splicing from one and the same gene, namely the MUC1 gene.

A genetic investigation of virulence in a rodent malaria parasite.

The genetic basis of virulence in a line (YM) of Plasmodium yoelii yoelii was investigated in a cross with a mild line (A/C). The blood forms of the virulent line developed extensively in mature erythrocytes of mice, causing death of the host within 7 days; infections with the mild line were normally restricted to reticulocytes, infected animals recovering after three weeks. Lines YM and A/C differed additionally in enzyme and drug-sensitivity markers. Studies on infections established from each line alone from sporozoite mixtures of the two lines and from the cross between the lines showed that the appearance of virulence had been caused by a genetic change in the parasite, and not by other factors such as a concurrent infection with another organism. An analysis of the characters of 56 clones derived from the cross showed that the virulence character had undergone recombination with the other markers, and appeared to be inherited in Mendelian fashion. Three clones exhibited atypical virulence, although it was not clear whether this had been produced by genetic recombination.

Immunological studies in rodent malaria. I: Protective immunity induced in mice by mild strains of Plasmodium berghei yoelii against a virulent and fatal line of this plasmodium.

Mild and virulent lines of Plasmodium berghei yoelii are readily distinguished both by their course of infection and by the preference of the virulent line for mature red blood cells. Mice given either mild line were fully protected against the virulent P.b. yoelii one week after they had become negative. Mice given the mild line of P.b. yoelii 17X were fully protected against a challenge by the virulent line on the third day of infection (D+3). Mice given the mild and virulent lines of P.b. yoelii 17X concomitantly showed only slight protection, all eventually succumbing. Mice given the virulent P.b. yoelii 17X and challenged with P.b. yoelii 17X mild on D+1 showed 2/6 with virulent-like infections and 4/6 infections which resembled the mild controls. Five out of eleven animals challenged with P.b. berghei after immunization with mild P.b. yoelii had infections which resembled the P.b. berghei control group and were dead by D+23. The other six mice became negative, but all of them relapsed and three of them subsequently died. In mice challenged four weeks after their mild P.b. yoelii 17X infections had become negative, 5/6 succumbed to a P.b. berghei infection. Mice immunized with P.b. yoelii 17X mild and challenged with P.v. vinckei one week after becoming negative had a survival rate of 2/10.

Effects of lowered environmental temperature on the growth of exoerythrocytic stages of Plasmodium berghei.

The effect of lowered host-environmental temperature upon the development and maturation of the preerythrocytic tissue stages of rodent malaria parasites has been investigated in two strains of Plasmodium berghei originating from the highlands of Katanga. Young albino rats inoculated with massive sporozoite doses of P. berghei NK 65 and maintained for 48 hours at 12 degrees C developed small, stunted tissue schizonts, averaging 11 X 15 microns, of a distinct morphology. Control rats kept at room temperature of 27 degrees C showed mature tissue schizonts of normal growth averaging 24 X 29 microns. Blood from the rats kept at lower temperature for 48 to 50 hours failed to produce parasitemia when inoculated into susceptible recipient mice. All the mice given blood from control rats developed parasitemia. However, when sporozite-inoculated rats were kept for 96 hours or longer at 12 degrees C they developed parasitemia and their liver showed maturation of 10% of the preerythrocytic schizonts. Experiments with the ANKA strain of P. berghei did not show significant differences in size and morphology between parasites in rats kept for 46.5 hours at 9 degrees C and 12 degrees C and those in controls kept at 20 degrees C. However, subinoculation of blood from the low temperature experimental groups into recipient mice at 46.5 hours after intravenous sporozoite inoculation failed to produce parasitemia, whereas all the recipient mice from the control groups developed parasitemia in 4 or 5 days. The findings are discussed in the light of the evolution of plasmodia and the phenomena of relapse and delayed primary attack in certain malaria infections.

Sudden increase in virulence in a strain of Plasmodium berghei yoelii.

The mild and chronic 17X strain of Plasmodium berghei yoelii showed a sudden increase in virulence following a period of 110 days in the deep freeze. The enhanced virulence was seen in a very high and early parasite peak in the blood and a 100% mortality of all infected mice. The exalted virulence remained unaltered following a number of blood transfers of the strain and after four cyclical transmissions through Anopheles stephensi. Enzyme pattern studies revealed that the virulent strain possessed the enzyme types GPI-1 and 6 PGD-4,-both characteristic for strain 17X of P.b. yoelii. Studies carried out to investigate the possibility of a concomitant viral infection transferred by blood passage, which could have been responsible for the enhanced virulence and mortality in the infected mice, showed no presence of any detectable virus in the blood of the P.b. yoelii infected mice. It is suggested that virulence may provide a marker in genetic work of rodent plasmodia and be included in association with enzyme and drug resistance markers.

Brain capillary blockage produced by a virulent strain of rodent malaria.

A sudden enhancement in virulence of a mild Plasmodium berghei yoelii 17 x strain resulted in fulminating and fatal infections in CF1 and A/J mice. The virulent strain has maintained its characteristics after ten cyclical transmissions through Anopheles stephensi. The visible expression of virulence of the mutated strain is its ability to cross the blood-brain barrier and cause intravascular sequestration of injected erythrocytes and blockage of brain capillaries. We, therfore, believe that the virulent line of Plasmodium berghei yoelii 17 x could serve as a useful laboratory model for the study of "cerebral malaria."