In Vitro Cell. Dev. Biol. 27A:185-187, March 1991 © 1991 Tissue Culture Association 0883-8364/91 $01.50+0.00
L e t t e r to t h e E d i t o r SUBPOPULATIONS OF COLON CANCER CELLS SURVIVE FREEZING
FP10 cells showed greater changes from FP5 cells. The P10 cells, while showing some further karyotypic evolution from P5, again remained predominantly (86%) derived from cell C. The FP10 cells showed chromosome evolution from cell B and as at FP5 each cell showed a distinct chromosomal constitution. The results confirm the differences for frozen and non-frozen cells seen at P5 and FP5. A feature of frozen cells not seen at levels P0, P5 or P10 was the presence of new structural (unbalanced) rearrangements, all involving a break in the terminal region of the derivative chromosome. P20 cells were not available for analysis, and the results are summarized in Table 1. Eight of 32 FP5 ceils showed unbalanced rearrangements; these were unique to individual cells in four cells and common to two cells in two further cases. Similarly, for our FP10 cells, 11 of 28 cells showed these structural changes: three cells showed the same rearrangements; in four cases the same translocation was seen in two different cells; and in the other four cases the rearrangements were unique. One FP5 cell showed the rearrangement (18;?) (q23;?) and three of 11 F P 1 0 cells had the same rearrangement: at passage level 20 (FP20) six of 28 ceils examined showed this same abnormality. Our results indicate a disparity of chromosome constitution for cells at passage levels, 0, 5 and 10 of continuous in vitro culture (P0, P5 and P10) and for cells frozen at - 1 3 0 ° C prior to passage of examination (FP5 and FP10). The diversification of chromosome content for tumor cell lines during in vitro culture has been observed by numerous researchers, and is most probably due to the selective measures imposed by the media and the conditions of culture (Fidler and Hart, 1982). However, a comparison of the P5 and P10 cells with those that had been frozen and examined at the same passage (FP5 and FP10) indicated a number of differences. Thus, chromosome numbers were more varied and there was an increasing tendency for cells with higher chromosome numbers to be present after freezing; karyotype analysis showed marked differences between P and PF cells; and unbalanced rearrangements were a feature of frozen but not unfrozen cells. The results indicated that freezing of ceils has a selective effect on the survival of cell populations, resulting in loss of certain clones; alternatively, the process may encourage more rapid diversification of karyotype constitution following reconstitution to in vitro culture. These effects should be considered when using cell lines to determine tumor cell properties: not only does in vitro passage result in diversification, but storing of cell lines at low temperatures may cause further distortion resulting in tumor cell lines even more remote from the original tumor cell population. The appearance of new unbalanced translocation, in each instant involving a terminal deletion is of interest, since these were not observed at any unfrozen passage levels. It may be related to the
Dear Editor: Tumor cell lines are usually preserved in liquid or vapor phase nitrogen under conditions which allow 8 0 - 9 0 % recovery of viable ceils: the assumption is made that the freezing process does not distort the spectrum of subpopulations of cells. We have tested this assumption using cells from an in vitro established mucinous colorectal tumor cell line NYT27 for which cytogenetic studies were carried out at passage levels 0, 5 and 10, (P0, P5 and P10) and on cells reconstituted from liquid nitrogen after in vitro passage level 4, 9 and 19 (FP5, FPIO and FP20). In particular, the influence of storage on new structural rearrangements was recorded. The methods of ceil culture and chromosome analysis of the NYT27 cells from the original tumor (PO) have been described (Yaseen et al., 1990). The distribution of chromosome numbers for 119 cells at passage five (P5) indicated significant differences from that of 100 FP5 cells. The numbers of chromosome in P5 ceils varied from 6 9 - 7 8 , with a regular distribution frequency between these extremes and a modal number of 7 4 - 7 5 ; in contrast, for FP5 cells, the chromosome number varied from 6 6 - 8 4 with a wide distribution frequency and a modal number of 7 4 - 7 8 . For P5 ceils, the karyotype of clone A was identified at P0, and its chromosome composition suggested that it was the stem clone from which other clones present at P5, were derived. Thirty-two P5 ceils were karyotyped; eight subclones were recognized, and the percentage distribution of each clone shown in Figure 1. Although subclones A, E, G, B and J identified at P0 were not present at P5, the major sublines D, E and F were present in a proportion similar to that seen at P0. The karyotype distribution of 22, FP5 cells is also shown in Figure 1. In this instance, although the cell karyotypes appeared to have been derived from B, showing trisomy #5, #11, #12, #21, no cells were identified with the t(17 ;21) seen in C which characterizes the karyotype evolution of both PO and P5; no two cells had the same chromosome complement, and all demonstrated a new evolution pattern from B. Thus, in both chromosome numbers, and in detailed karyotype characteristics, P5 and FP5 cells showed marked differences. After 10 in vitro passages of cell line NYT27 (P 10), the distribution of chromosome numbers for 100 cells varied from 69 to 82 without a regular distribution curve or model number; however, more cells with a higher number of chromosomes were identified than at P5. In contrast, similar analysis for 100 cells frozen at passage nine and reconstituted (FP10) showed chromosome numbers varying from 67 to 84 with a model number of 78 to 79. In comparison with P10 cells, FP10 contained more cells with a higher number of chromosomes. Thirty-six P10 ceils and 28 FP10 cells karyotyped: the results were not significantly different from that of P5 and FP5 cells; however, P10 cells showed increased diversity in chromosome number when compared to P5 cells, whilst 185
186
YASEEN ET AL.
PROPOSED KARYOTYPE DERIVATION OF NYT27 CELLS Passage P5
Passage FP5
) +2 +O
C)
FIe. 1. Karyotypes in NYT27 cells after 5 in vitro passages (P5) or 4 passages in vitro followed by freezing and re-constitution (FP5). © - - clones present on an original tumor cell population, but not seen at P5 or FP5. For P5 cells, the percentage of each clone in the total population is given.
187
CELL POPULATIONS AFTER FREEZING
would be consistent with the present findings. The significance of translocations in not known: however, the prevalence of these structural rearrangements in cells after freezing suggest that the process of freezing could be an active trigger to this process when cells are subsequently reconstituted.
TABLE 1 THE FREQUENCY AND NATURE OF STRUCTURAL REARRANGEMENTS IN POST-FREEZING PASSAGES OF THE NYT27 CELL LINE Passage No. (No. of Cells Examined)
ChromosomesInvolvedin Terminal Rearrangement(TR)
No. of Cells REFERENCES
FP5 (32)
(6;7) (X;X) (X;X) (6;20) (18;?) (10;10)
(q27;q26) (p22;q28) (p22;p22) (p25:q13) (q23;?) (p22;q28)
1 1 1 2 1 2
FP10 (28)
(10;16) (6;10) (10;12) (6;7) (18;?) (11;?) (X;X)
(p15;p13) (q27;plS) (p15;q24) (q27;p22) (q23;?) (p15;?) (p22;q28)
2 1 1 2 3 1 1
FP20 (28)
(X;10) (X;10) (7;11) (2;?) (14;?) (19;?) (18;?)
(q28;p15) (p22;p15) (q36;p15) (q37;?) (p13;?) (q13;?) (q23;?)
2 2 1 1 1 2 6
formation of terminal rearrangements which have been described in human meningiomas (Mahby et al., 1988), B cell leukaemia (Fitzgerald and Morris, 1984) and renal tumors (Kovacs et al., 1987). Saltman et al. (1989) demonstrated the appearance of such translocations in the chromosomes of some subclones of human lymphoblastic cell lines after six weeks in culture, but the authors did not indicate whether the cell line had been frozen during the study; indeed, if freeze/thawing was introduced at some point in the passage of the tumor cells quoted above the presence of translocations
Fidler, I. J.; Hart, I. R. Biologicaldiversity in metastatic neoplasms: origins and implications. Science 217:998-1003; 1982. Fitzgerald, P. H.; Morris, C. M. Telomeric association of chromosomes in B-cell lymphoid leukemia. Human Genet. 67:385-390; 1984. Kovacs, G.; Muller-Brechlin, R.; Szucs, S. Telomefic association in two human renal tumours. Cancer Genet. Cytogenet. 28:363-366; 1987. Maltby. E. L.; lronside, J. W.; Battersby, R. D. E. Cytogeneticstudies in 50 meningiomas. Cancer Genet. Cytogenet. 31:199-210; 1988. Sahman, D.; Ross, F. M.; Fantes, J. A., et al. Telomeric association in a lymphoblastoid cell line from a patient with B-cell follicular lymphoma. Cytogenet. Cell Genet. 50:230-233; 1989. Yaseen, N. Y.; Watmore, A. E.; Potter, A. M.. el al. Chromosome studies in eleven colorectal tumours. Cancer Genet. Cytogenet. 44:83-87; 1990. N. Y. Yaseen A. E. Watmore A. M. Potter
C.W. Potter G. Jacob R.C. Rees
Section of Tumor Biology and Immunology, Department of Experimental and Clinical Microbiology (N. Y. Y., C. W. P., R. C. R.), Department of Medical Genetics (A. E. W., A. M. P.) and Surgery (G. J.) University of Sheffield Medical School, Sheffield, SIO 2RX, S. Yorkshire, England (Received 16 July 1990)
This work is supported by the Yorkshire Cancer Research Campaign.
L e t t e r to t h e E d i t o r SUBPOPULATIONS OF COLON CANCER CELLS SURVIVE FREEZING
FP10 cells showed greater changes from FP5 cells. The P10 cells, while showing some further karyotypic evolution from P5, again remained predominantly (86%) derived from cell C. The FP10 cells showed chromosome evolution from cell B and as at FP5 each cell showed a distinct chromosomal constitution. The results confirm the differences for frozen and non-frozen cells seen at P5 and FP5. A feature of frozen cells not seen at levels P0, P5 or P10 was the presence of new structural (unbalanced) rearrangements, all involving a break in the terminal region of the derivative chromosome. P20 cells were not available for analysis, and the results are summarized in Table 1. Eight of 32 FP5 ceils showed unbalanced rearrangements; these were unique to individual cells in four cells and common to two cells in two further cases. Similarly, for our FP10 cells, 11 of 28 cells showed these structural changes: three cells showed the same rearrangements; in four cases the same translocation was seen in two different cells; and in the other four cases the rearrangements were unique. One FP5 cell showed the rearrangement (18;?) (q23;?) and three of 11 F P 1 0 cells had the same rearrangement: at passage level 20 (FP20) six of 28 ceils examined showed this same abnormality. Our results indicate a disparity of chromosome constitution for cells at passage levels, 0, 5 and 10 of continuous in vitro culture (P0, P5 and P10) and for cells frozen at - 1 3 0 ° C prior to passage of examination (FP5 and FP10). The diversification of chromosome content for tumor cell lines during in vitro culture has been observed by numerous researchers, and is most probably due to the selective measures imposed by the media and the conditions of culture (Fidler and Hart, 1982). However, a comparison of the P5 and P10 cells with those that had been frozen and examined at the same passage (FP5 and FP10) indicated a number of differences. Thus, chromosome numbers were more varied and there was an increasing tendency for cells with higher chromosome numbers to be present after freezing; karyotype analysis showed marked differences between P and PF cells; and unbalanced rearrangements were a feature of frozen but not unfrozen cells. The results indicated that freezing of ceils has a selective effect on the survival of cell populations, resulting in loss of certain clones; alternatively, the process may encourage more rapid diversification of karyotype constitution following reconstitution to in vitro culture. These effects should be considered when using cell lines to determine tumor cell properties: not only does in vitro passage result in diversification, but storing of cell lines at low temperatures may cause further distortion resulting in tumor cell lines even more remote from the original tumor cell population. The appearance of new unbalanced translocation, in each instant involving a terminal deletion is of interest, since these were not observed at any unfrozen passage levels. It may be related to the
Dear Editor: Tumor cell lines are usually preserved in liquid or vapor phase nitrogen under conditions which allow 8 0 - 9 0 % recovery of viable ceils: the assumption is made that the freezing process does not distort the spectrum of subpopulations of cells. We have tested this assumption using cells from an in vitro established mucinous colorectal tumor cell line NYT27 for which cytogenetic studies were carried out at passage levels 0, 5 and 10, (P0, P5 and P10) and on cells reconstituted from liquid nitrogen after in vitro passage level 4, 9 and 19 (FP5, FPIO and FP20). In particular, the influence of storage on new structural rearrangements was recorded. The methods of ceil culture and chromosome analysis of the NYT27 cells from the original tumor (PO) have been described (Yaseen et al., 1990). The distribution of chromosome numbers for 119 cells at passage five (P5) indicated significant differences from that of 100 FP5 cells. The numbers of chromosome in P5 ceils varied from 6 9 - 7 8 , with a regular distribution frequency between these extremes and a modal number of 7 4 - 7 5 ; in contrast, for FP5 cells, the chromosome number varied from 6 6 - 8 4 with a wide distribution frequency and a modal number of 7 4 - 7 8 . For P5 ceils, the karyotype of clone A was identified at P0, and its chromosome composition suggested that it was the stem clone from which other clones present at P5, were derived. Thirty-two P5 ceils were karyotyped; eight subclones were recognized, and the percentage distribution of each clone shown in Figure 1. Although subclones A, E, G, B and J identified at P0 were not present at P5, the major sublines D, E and F were present in a proportion similar to that seen at P0. The karyotype distribution of 22, FP5 cells is also shown in Figure 1. In this instance, although the cell karyotypes appeared to have been derived from B, showing trisomy #5, #11, #12, #21, no cells were identified with the t(17 ;21) seen in C which characterizes the karyotype evolution of both PO and P5; no two cells had the same chromosome complement, and all demonstrated a new evolution pattern from B. Thus, in both chromosome numbers, and in detailed karyotype characteristics, P5 and FP5 cells showed marked differences. After 10 in vitro passages of cell line NYT27 (P 10), the distribution of chromosome numbers for 100 cells varied from 69 to 82 without a regular distribution curve or model number; however, more cells with a higher number of chromosomes were identified than at P5. In contrast, similar analysis for 100 cells frozen at passage nine and reconstituted (FP10) showed chromosome numbers varying from 67 to 84 with a model number of 78 to 79. In comparison with P10 cells, FP10 contained more cells with a higher number of chromosomes. Thirty-six P10 ceils and 28 FP10 cells karyotyped: the results were not significantly different from that of P5 and FP5 cells; however, P10 cells showed increased diversity in chromosome number when compared to P5 cells, whilst 185
186
YASEEN ET AL.
PROPOSED KARYOTYPE DERIVATION OF NYT27 CELLS Passage P5
Passage FP5
) +2 +O
C)
FIe. 1. Karyotypes in NYT27 cells after 5 in vitro passages (P5) or 4 passages in vitro followed by freezing and re-constitution (FP5). © - - clones present on an original tumor cell population, but not seen at P5 or FP5. For P5 cells, the percentage of each clone in the total population is given.
187
CELL POPULATIONS AFTER FREEZING
would be consistent with the present findings. The significance of translocations in not known: however, the prevalence of these structural rearrangements in cells after freezing suggest that the process of freezing could be an active trigger to this process when cells are subsequently reconstituted.
TABLE 1 THE FREQUENCY AND NATURE OF STRUCTURAL REARRANGEMENTS IN POST-FREEZING PASSAGES OF THE NYT27 CELL LINE Passage No. (No. of Cells Examined)
ChromosomesInvolvedin Terminal Rearrangement(TR)
No. of Cells REFERENCES
FP5 (32)
(6;7) (X;X) (X;X) (6;20) (18;?) (10;10)
(q27;q26) (p22;q28) (p22;p22) (p25:q13) (q23;?) (p22;q28)
1 1 1 2 1 2
FP10 (28)
(10;16) (6;10) (10;12) (6;7) (18;?) (11;?) (X;X)
(p15;p13) (q27;plS) (p15;q24) (q27;p22) (q23;?) (p15;?) (p22;q28)
2 1 1 2 3 1 1
FP20 (28)
(X;10) (X;10) (7;11) (2;?) (14;?) (19;?) (18;?)
(q28;p15) (p22;p15) (q36;p15) (q37;?) (p13;?) (q13;?) (q23;?)
2 2 1 1 1 2 6
formation of terminal rearrangements which have been described in human meningiomas (Mahby et al., 1988), B cell leukaemia (Fitzgerald and Morris, 1984) and renal tumors (Kovacs et al., 1987). Saltman et al. (1989) demonstrated the appearance of such translocations in the chromosomes of some subclones of human lymphoblastic cell lines after six weeks in culture, but the authors did not indicate whether the cell line had been frozen during the study; indeed, if freeze/thawing was introduced at some point in the passage of the tumor cells quoted above the presence of translocations
Fidler, I. J.; Hart, I. R. Biologicaldiversity in metastatic neoplasms: origins and implications. Science 217:998-1003; 1982. Fitzgerald, P. H.; Morris, C. M. Telomeric association of chromosomes in B-cell lymphoid leukemia. Human Genet. 67:385-390; 1984. Kovacs, G.; Muller-Brechlin, R.; Szucs, S. Telomefic association in two human renal tumours. Cancer Genet. Cytogenet. 28:363-366; 1987. Maltby. E. L.; lronside, J. W.; Battersby, R. D. E. Cytogeneticstudies in 50 meningiomas. Cancer Genet. Cytogenet. 31:199-210; 1988. Sahman, D.; Ross, F. M.; Fantes, J. A., et al. Telomeric association in a lymphoblastoid cell line from a patient with B-cell follicular lymphoma. Cytogenet. Cell Genet. 50:230-233; 1989. Yaseen, N. Y.; Watmore, A. E.; Potter, A. M.. el al. Chromosome studies in eleven colorectal tumours. Cancer Genet. Cytogenet. 44:83-87; 1990. N. Y. Yaseen A. E. Watmore A. M. Potter
C.W. Potter G. Jacob R.C. Rees
Section of Tumor Biology and Immunology, Department of Experimental and Clinical Microbiology (N. Y. Y., C. W. P., R. C. R.), Department of Medical Genetics (A. E. W., A. M. P.) and Surgery (G. J.) University of Sheffield Medical School, Sheffield, SIO 2RX, S. Yorkshire, England (Received 16 July 1990)
This work is supported by the Yorkshire Cancer Research Campaign.
