Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells

Abstract

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

Figure 1

Retroviral transduction with tsLT and hTERT. (A) The U19tsA58 LT virus (27) was constructed by using the pZipNeoSV(X)1 backbone and encodes a full-length LT cDNA from U19tsA58 and confers resistance to G418. The U19tsA58 LT is a combination of two mutants, U19, which encodes a LT that does not bind specifically to SV40-origin DNA sequences, and tsA58, which encodes a thermolabile LT antigen that is wild type at 33.5°C but inactive at 39.5°C. (B) The hTERT virus was constructed by using the pBabe–hygro backbone and encodes a full-length hTERT cDNA as well as the hygromycin resistance gene. (C) Sequence of retroviral gene transduction and resulting population-doubling (PD) potential of human adult mammary fibroblasts from a single 19-year-old donor (HMF3).

Figure 2

Telomerase activity. (A) 20- and 200-ng aliquots of cellular extracts were assayed for the presence of telomerase activity by the telomeric repeat amplification protocol (TRAP) assay. Extracts (200 ng each) were heat-treated (HI) to inactivate telomerase and assayed as a negative control. An internal PCR standard was included in each PCR to demonstrate that an absence of telomerase did not result from PCR inhibitors in the cell extracts. (B) Telomerase activity measured by the TRAP assay was detected in cellular extracts (20 and 200 ng) from HMF3 and HMME7 cells containing ectopic hTERT and U19tsA58 LT cultured at both 33.5°C (permissive temperature for tsA58 LT) and at 39.5°C (nonpermissive temperature) for 7 days. Activity was not detected in HMF3 and HMME7 cells containing U19tsA58 LT alone. (C) Southern blot showing TRF lengths for the fibroblast cultures HMF3 at p3 (lane 1), p12 (lane 2), p18 (lane 3), p24 after early transduction of tsLT (lane 4), p23 after late transduction of tsLT (lane 5), and p17 after both early (lane 6) and late (lane 7) transduction of hTERT. Numerical values for mean TRF lengths of these and other cultures are shown in Table 1.

Figure 3

Effects of U19tsA58 LT inactivation on growth. (A) Growth of HMF3A, -B, -C and -D at late passage (>p30) compared with that of untransduced cells (control 3 at p10) and cells transduced with only the tsLT gene (tsLT3 at p18). Cells were grown at permissive (33.5°C; open bars), semipermissive (36.5°C; hatched bars), and nonpermissive (39.5°C; cross-hatched bars) temperatures. Relative growth rates (means ± SD, n = 3) are expressed as a percentage of cell numbers at permissive temperature. (B) Growth of HMME2 and HMME7 at late passage (>p30) compared with that of corresponding untransduced controls and tsLT-only transduced cells at 33.5°C (open bars), 36.5°C (hatched bars), and 39.5°C (cross-hatched bars). Relative growth rates (means ± SD, n = 3) are expressed as a percentage of cell numbers at permissive temperature.

Figure 4

Cell-cycle analysis. Flow cytograms showing cell-cycle parameters of HMME7 (A and B), HMF3A (C and D), and HMF3B (E and F). Cells were grown at either 33.5°C (A, C, and E) or 39.5°C (B, D, and F) for 5 days and pulse labeled with BrdUrd. Plots show PI fluorescence (x axis) vs. FITC fluorescence from BrdUrd (y axis). The proportion of cells in S phase is shown, being reduced to nearly zero in HMME7 and HMF3A but not HMF3B cells, at 39.5°C.

Figure 5

Clonal analysis of growth arrest. (A) Relative CE of the four fibroblast cultures at 33.5°C after 0 days (open bars), 3 days (▨), 7 days (cross-hatched bars), 10 days (▧), and 14 days (solid bars) at 39.5°C. Results are means of duplicate determinations (variance of replicate CE < ± 10%). (B) Relative colony-forming efficiency at 33.5°C of the endothelial culture HMME7 after 0 days (open bars), 3 days (▨), 6 days (cross-hatched bars), and 9 days (▧) at 36.5°C and at 39.5°C, respectively. Results are means ± SD (n = 4). The corresponding numbers of SA β-galactosidase positive cells were 5% (day 0), 11% (day 3), 54% (day 6), and 79% (day 9) in cultures at 36.5°C, compared with 3% (day 3), 6% (day 6), and 12% (day 9) in cultures at 39.5°C.